U.S. patent application number 15/916986 was filed with the patent office on 2018-09-20 for fused pyrimidine-based hydroxamate derivatives.
The applicant listed for this patent is Agency for Science, Technology and Research. Invention is credited to Dizhong Chen, Chang Kai Soh, Haishan Wang.
Application Number | 20180265512 15/916986 |
Document ID | / |
Family ID | 54072177 |
Filed Date | 2018-09-20 |
United States Patent
Application |
20180265512 |
Kind Code |
A1 |
Wang; Haishan ; et
al. |
September 20, 2018 |
FUSED PYRIMIDINE-BASED HYDROXAMATE DERIVATIVES
Abstract
The present invention relates to fused pyrimidine-based
hydroxamate compounds of formula (I), comprising a hydroxamate
group, that are inhibitors of hiStone deacetylase (HDAC) and
kinases. More particularly, the present invention relates to
hydroxamate substituted purine or 5H-pyrrolo[3,2-d]pyrimidine
derivatives, methods for their preparation, pharmaceutical
compositions containing these compounds and uses of these compounds
in the treatment of disorders/conditions/diseases involving,
relating to or associated with enzymes having histone deacetylase,
non-histone deacetylase and kinase activities/functions and/or via
unspecified/multi-targeted mechanisms.
Inventors: |
Wang; Haishan; (Singapore,
SG) ; Chen; Dizhong; (Singapore, SG) ; Soh;
Chang Kai; (Singapore, SG) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Agency for Science, Technology and Research |
Singapore |
|
SG |
|
|
Family ID: |
54072177 |
Appl. No.: |
15/916986 |
Filed: |
March 9, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15124654 |
Sep 8, 2016 |
9957270 |
|
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PCT/SG2015/050038 |
Mar 13, 2015 |
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15916986 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 7/00 20180101; A61P
11/06 20180101; A61P 37/08 20180101; A61P 27/02 20180101; A61K
31/52 20130101; A61P 29/00 20180101; A61P 1/16 20180101; A61P 37/00
20180101; A61P 35/00 20180101; A61K 45/06 20130101; A61P 35/02
20180101; C07D 473/34 20130101; A61P 31/18 20180101; C07D 473/40
20130101; A61P 25/00 20180101; A61P 25/28 20180101; C07D 473/16
20130101; A61K 31/5377 20130101; A61P 43/00 20180101; A61P 37/02
20180101; A61P 13/12 20180101; A61P 25/14 20180101; A61P 21/00
20180101; C07D 473/32 20130101; A61K 31/5377 20130101; A61K 2300/00
20130101; A61K 31/52 20130101; A61K 2300/00 20130101 |
International
Class: |
C07D 473/34 20060101
C07D473/34; A61K 31/52 20060101 A61K031/52; C07D 473/40 20060101
C07D473/40; A61K 45/06 20060101 A61K045/06; A61K 31/5377 20060101
A61K031/5377; C07D 473/32 20060101 C07D473/32; C07D 473/16 20060101
C07D473/16 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 13, 2014 |
SG |
10201400634Q |
Claims
1.-45. (canceled)
46. A method of inhibiting HDAC and/or PI3K in a cell comprising
administering to a cell a compound of Formula (I), ##STR00152##
wherein X, Y and Z are independently selected from N, CHR.sup.3 or
CR.sup.3, wherein at least one of X, Y or Z is N; is a single or
double bond, as valency allows; R.sup.1 and R.sup.2 are
independently selected from the group consisting of a bond,
halogen, optionally substituted alkyl, optionally substituted
amino, optionally substituted alkyloxy, optionally substituted
cycloalkyl, optionally substituted heterocycloalkyl, optionally
substituted aryl and optionally substituted heteroaryl; R.sup.3 and
R.sup.4 are independently selected from the group consisting of a
bond, hydrogen, halogen, optionally substituted alkyl, optionally
substituted amino, optionally substituted alkyloxy, optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl and optionally substituted heteroaryl;
at least one of R.sup.1, R.sup.2, R.sup.3 or R.sup.4 is further
independently substituted by an hydroxamate group
-L.sup.1-R.sup.5-L.sup.2-R.sup.6-L.sup.3-CON(R.sup.a)OR.sup.b,
wherein; R.sup.a and R.sup.b are independently selected from the
group consisting of a bond, hydrogen, optionally substituted alkyl,
optionally substituted acyl and optionally substituted amino acid
residue; L.sup.1, L.sup.2 and L.sup.3 are independently selected
from the group consisting of a bond, optionally substituted alkyl,
optionally substituted alkenyl and optionally substituted alkynyl;
R.sup.5 and R.sup.6 are independently selected from the group
consisting of a bond, O, S, NR.sup.c, S(O).sub.n, optionally
substituted amide, optionally substituted urea, optionally
substituted carbonylurea, optionally substituted thiourea,
optionally substituted sulfonamide, optionally substituted
aminosulfonamide, optionally substituted sulfonylurea, optionally
substituted oxime, optionally substituted cycloalkyl, optionally
substituted heterocycloalkyl, optionally substituted aryl and
optionally substituted heteroaryl; wherein; R.sup.c is
independently selected from the group consisting of a bond,
hydrogen, optionally substituted alkyl, optionally substituted
alkenyl, optionally substituted alkynyl and optionally substituted
acyl; and n is an integer from 0 to 2; or a pharmaceutically
acceptable form or prodrug thereof.
47. The method according to claim 46, wherein the inhibition of
HDAC and/or PI3K further comprises the inhibition of cell
proliferation or reprogramming cells to induce pluripotent stem
cells (iPS cells), wherein the cell is in vitro or the cell is from
tissue of a subject or the cell is in a subject.
48. (canceled)
49. (canceled)
50. The method according to claim 47, wherein the cell is from a
cell line, wherein the cell line is an immortalized cell line, a
genetically modified cell line or a primary cell line, or is
selected from the group consisting of MV4-11, MOLT-4, PC-3, MCF-7,
SUP-B15, HL-60, K-562, RPMI-8226, Daudi, Raji, Ramos, Pfeiffer,
A431, ACHN, A549, COLO 205, HCT116, HEL92.1.7, NCI-H522, A375,
NCI-H460, BxPC-3, PANC-1, SK-OV-3, U87MG, U138MG, HpeG2, SK-HEP1,
HuH-7, HCCLM3, PLC/PRF/5, HeLa, BT 474, MDA-MB-231, MDA-MB-436 and
MDA-MB-468.
51.-54. (canceled)
55. A method of treating a HDAC- and/or PI3K-related disorder
comprising administering to a subject in need of treatment a
compound of Formula (I), ##STR00153## wherein X, Y and Z are
independently selected from N, CHR.sup.3 or CR.sup.3, wherein at
least one of X, Y or Z is N; is a single or double bond, as valency
allows; R.sup.1 and R.sup.2 are independently selected from the
group consisting of a bond, halogen, optionally substituted alkyl,
optionally substituted amino, optionally substituted alkyloxy,
optionally substituted cycloalkyl, optionally substituted
heterocycloalkyl, optionally substituted aryl and optionally
substituted heteroaryl; R.sup.3 and R.sup.4 are independently
selected from the group consisting of a bond, hydrogen, halogen,
optionally substituted alkyl, optionally substituted amino,
optionally substituted alkyloxy, optionally substituted cycloalkyl,
optionally substituted heterocycloalkyl, optionally substituted
aryl and optionally substituted heteroaryl; at least one of
R.sup.1, R.sup.2, R.sup.3 or R.sup.4 is further independently
substituted by an hydroxamate group
-L.sup.1-R.sup.5-L.sup.2-R.sup.6-L.sup.3-CON(R.sup.a)OR.sup.b,
wherein; R.sup.a and R.sup.b are independently selected from the
group consisting of a bond, hydrogen, optionally substituted alkyl,
optionally substituted acyl and optionally substituted amino acid
residue; L.sup.1, L.sup.2 and L.sup.3 are independently selected
from the group consisting of a bond, optionally substituted alkyl,
optionally substituted alkenyl and optionally substituted alkynyl;
R.sup.5 and R.sup.6 are independently selected from the group
consisting of a bond, O, S, NR.sup.c, S(O).sub.n, optionally
substituted amide, optionally substituted urea, optionally
substituted carbonylurea, optionally substituted thiourea,
optionally substituted sulfonamide, optionally substituted
aminosulfonamide, optionally substituted sulfonylurea, optionally
substituted oxime, optionally substituted cycloalkyl, optionally
substituted heterocycloalkyl, optionally substituted aryl and
optionally substituted heteroaryl; wherein; R.sup.c is
independently selected from the group consisting of a bond,
hydrogen, optionally substituted alkyl, optionally substituted
alkenyl, optionally substituted alkynyl and optionally substituted
acyl; and n is an integer from 0 to 2, or a pharmaceutically
acceptable form or prodrug thereof, or a composition comprising a
compound of Formula (I), or a pharmaceutically acceptable form or
prodrug thereof, and a pharmaceutically acceptable excipient.
56. (canceled)
57. The method according to claim 55, wherein the disorder is
cancer, angiogenic disorder or pathological angiogenesis, fibrosis,
inflammatory conditions, asthma, neurological disorders,
neurodegenerative disorders, muscle degenerative disorders,
autoimmune disorders, disorders of the blood or disorders of the
bone marrow, or is lymphoma, cutaneous T-cell lymphoma, follicular
lymphoma, or Hodgkin lymphoma, cervical cancer, ovarian cancer,
breast cancer, lung cancer, prostate cancer, colorectal cancer,
sarcoma, hepatocellular carcinoma, leukemia or myeloma, retinal
angiogenic disease, liver fibrosis, kidney fibrosis, Alzheimer's
disease or Huntington's disease, spinal muscular atrophy, HIV/AIDS,
polycythemia vera or essential thrombocythemia or
myelofibrosis.
58. (canceled)
59. (canceled)
60. A method of modulating the self-renewal or differentiation of
stem-cells comprising administering to a subject in need of
treatment a compound of Formula (I), ##STR00154## wherein X, Y and
Z are independently selected from N, CHR.sup.3 or CR.sup.3, wherein
at least one of X, Y or Z is N; is a single or double bond, as
valency allows; R.sup.1 and R.sup.2 are independently selected from
the group consisting of a bond, halogen, optionally substituted
alkyl, optionally substituted amino, optionally substituted
alkyloxy, optionally substituted cycloalkyl, optionally substituted
heterocycloalkyl, optionally substituted aryl and optionally
substituted heteroaryl; R.sup.3 and R.sup.4 are independently
selected from the group consisting of a bond, hydrogen, halogen,
optionally substituted alkyl, optionally substituted amino,
optionally substituted alkyloxy, optionally substituted cycloalkyl,
optionally substituted heterocycloalkyl, optionally substituted
aryl and optionally substituted heteroaryl; at least one of
R.sup.1, R.sup.2, R.sup.3 or R.sup.4 is further independently
substituted by an hydroxamate group
-L.sup.1-R.sup.5-L.sup.2-R.sup.6-L.sup.3-CON(R.sup.a)OR.sup.b,
wherein; R.sup.a and R.sup.b are independently selected from the
group consisting of a bond, hydrogen, optionally substituted alkyl,
optionally substituted acyl and optionally substituted amino acid
residue; L.sup.1, L.sup.2 and L.sup.3 are independently selected
from the group consisting of a bond, optionally substituted alkyl,
optionally substituted alkenyl and optionally substituted alkynyl;
R.sup.5 and R.sup.6 are independently selected from the group
consisting of a bond, O, S, NR.sup.c, S(O).sub.n, optionally
substituted amide, optionally substituted urea, optionally
substituted carbonylurea, optionally substituted thiourea,
optionally substituted sulfonamide, optionally substituted
aminosulfonamide, optionally substituted sulfonylurea, optionally
substituted oxime, optionally substituted cycloalkyl, optionally
substituted heterocycloalkyl, optionally substituted aryl and
optionally substituted heteroaryl; wherein; R.sup.c is
independently selected from the group consisting of a bond,
hydrogen, optionally substituted alkyl, optionally substituted
alkenyl, optionally substituted alkynyl and optionally substituted
acyl; and n is an integer from 0 to 2; or a pharmaceutically
acceptable form or prodrug thereof, or a composition comprising a
compound of Formula (I), or a pharmaceutically acceptable form or
prodrug thereof, and a pharmaceutically acceptable excipient.
61.-71. (canceled)
Description
TECHNICAL FIELD
[0001] The present invention generally relates to fused
pyrimidine-based hydroxamate compounds that are inhibitors of
histone deacetylase (HDAC) and kinases. More particularly, the
present invention relates to hydroxamate substituted purine or
5H-pyrrolo[3,2-d]pyrimidine derivatives, methods for their
preparation, pharmaceutical compositions containing these compounds
and uses of these compounds in the treatment of
disorders/conditions/diseases involving, relating to or associated
with enzymes having histone deacetylase, non-histone deacetylase
and kinase activities.
BACKGROUND ART
[0002] There is a general interest in the design, synthesis and
development of hybrid-drugs or multi-target drugs which can
increase the probability of the treatment or efficacy by acting on
two or more proven pathways of validated targets. For example,
cancer cell survival relies on many key pathways, thus, the
blockade or inhibition of one pathway may only have a small
probability of killing the cancer cells or inhibiting the growth of
cancer cells. Cancer cells can compensate or bypass the blocked
function or pathway and even synergize their functions. This
principle has been validated and is already in use, for example, in
combination chemotherapy for cancer treatment, where a combination
of drugs such as histone deacetylase (HDAC) inhibitors vorinostat
and a variety of known drugs in clinical trials, cocktail drugs for
HIV treatment, and augmentin (a mixture of amoxicillin and
clavulanic acid) for antibacterial treatment are administered
together. There are also successful multi-target drugs in the
market, such as multi-kinase inhibitors sunitinib and sorafenib.
Both combination therapy and multi-target drugs are aimed to
enhance efficacy and/or overcome drug resistance by modulating or
inhibiting multiple targets, pathways or networks involved in
disease progression. Combination therapy or polytherapy uses more
than one drug, thus its advantage is that there are a number of
single agents that are available for combination and there are
options for testing a variety of combinations of drugs for research
and development. However, combination therapy has disadvantages.
For example, single agents in general are developed for single
agent therapy only and are not necessarily optimized for
combination therapy. Further, not all single agents are suitable
for or compatible with combination therapy. Defining the dosage
regime for two or more agents in combination is a very complex
process which requires consideration for dosage level, sequence of
administration and potential drug-drug interaction in clinical
settings. Further, in addition to the cost of having to use
multiple drugs, combination therapy can often lead to unwanted
adverse effects or dangerous drug-drug interactions. For example,
everolimus was combined with sorafenib in a phase I clinical trials
for treatment of advanced hepatocellular carcinoma cancer (HCC),
but its dose could not be escalated to a biologically effective
concentration due to adverse events.
[0003] In contrast, a multi-target drug molecule, as a single
agent, works on at least two targets. The advantage of multi-target
drugs is that a single agent can achieve modulation of multiple
(kinase) targets simultaneously. However, the number of drugs that
can do this is still limited. As multi-target drugs typically
encompass the chemical features of the scaffolds of both parent
drugs, the molecular weight or size of the drug is usually larger,
and this often leads to the drug not receiving sufficient exposure
either due to toxicity or drug metabolism. It is not a trivial task
to design new molecules based on two scaffolds of the parent drugs.
Usually the desired efficacy is not obtained or there are new
undesired side effects. It is therefore not predictable how to
combine two scaffolds to achieve a new multi-target drug. Therefore
an observed good activity without undesired side effects is a
surprising finding.
[0004] There is therefore a need to provide a compound that
overcomes, or at least ameliorates, one or more of the
disadvantages described above. There is also a need to provide a
pharmaceutical composition comprising the compound, methods for
treating diseases using the compound and a method for synthesizing
the compound.
SUMMARY
[0005] A series of fused pyrimidine-based small molecules to target
histone deacetylases (HDACs) and phosphatidylinositide 3-kinases
(PI3K)-AKT-mammalian target of rapamycin (mTOR) pathway have been
designed and synthesised. These molecules contain a zinc-binding
group (hydroxamic acid) to inhibit histone deacetylase and other
deacetylase activities as well as a fused pyrimidine core decorated
with substitutes to modulate PI3K-AKT-mTOR pathway. Each molecule
has a unique potency profile against each targets and the entire
series covers most of possible combinations of the broad range of
potency required for each target for a variety of indications or
applications. These molecules work as multi-target drugs for
treatment of cancer and non-oncology applications.
[0006] In a first aspect, there is provided a compound of formula
(I);
##STR00001##
[0007] wherein X, Y and Z are independently selected from N,
CHR.sup.3 or CR.sup.3, wherein at least one of X, Y or Z is N; is a
single or double bond, as valency allows; R.sup.1 and R.sup.2 are
independently selected from the group consisting of a bond,
halogen, optionally substituted alkyl, optionally substituted
amino, optionally substituted alkyloxy, optionally substituted
cycloalkyl, optionally substituted heterocycloalkyl, optionally
substituted aryl and optionally substituted heteroaryl; R.sup.3 and
R.sup.4 are independently selected from the group consisting of a
bond, hydrogen, halogen, optionally substituted alkyl, optionally
substituted amino, optionally substituted alkyloxy, optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl and optionally substituted heteroaryl;
at least one of R.sup.1, R.sup.2, R.sup.3 or R.sup.4 is further
independently substituted by an hydroxamate group
-L.sup.1-R.sup.5-L-R.sup.6-L.sup.3-CON(R.sup.a)OR.sup.b, wherein;
R.sup.a and R.sup.b are independently selected from the group
consisting of a bond, hydrogen, optionally substituted alkyl,
optionally substituted acyl and optionally substituted amino acid
residue; L.sup.1, L.sup.2 and L.sup.3 are independently selected
from the group consisting of a bond, optionally substituted alkyl,
optionally substituted alkenyl and optionally substituted alkynyl;
R.sup.5 and R.sup.6 are independently selected from the group
consisting of a bond, O, S, NR.sup.c, S(O).sub.1, optionally
substituted amide, optionally substituted urea, optionally
substituted carbonylurea, optionally substituted thiourea,
optionally substituted sulfonamide, optionally substituted
aminosulfonamide, optionally substituted sulfonylurea, optionally
substituted oxime, optionally substituted cycloalkyl, optionally
substituted heterocycloalkyl, optionally substituted aryl and
optionally substituted heteroaryl; wherein; R.sup.c is
independently selected from the group consisting of a bond,
hydrogen, optionally substituted alkyl, optionally substituted
alkenyl, optionally substituted alkynyl and optionally substituted
acyl; and n is an integer from 0 to 2; or a pharmaceutically
acceptable form or prodrug thereof.
[0008] In a second aspect, there is provided a pharmaceutical
composition comprising a compound as defined above, or a
pharmaceutically acceptable form or prodrug thereof, and a
pharmaceutically acceptable excipient.
[0009] In further embodiments of the disclosure, a method of
inhibiting a deacetylase and/or kinase selected from the group
consisting of a lipid kinase/protein kinase or a fragment or a
complex thereof or a functional equivalent thereof and a PI3K or
Akt kinase or mTOR kinase or a fragment or a complex thereof or a
functional equivalent thereof, the method including exposing the
protein kinase or a fragment or complex thereof or a functional
equivalent thereof and/or co-factor(s) thereof to an effective
amount of a compound as defined above, is disclosed.
[0010] In some embodiments, the deacetylase is a histone
deacetylase or a fragment or a complex thereof or a functional
equivalent thereof. In some embodiments the histone deacetylase or
a fragment or complex thereof is HDAC1 or HDAC2 or HDAC3 or HDAC6
or HDAC8 or a fragment thereof, or a complex thereof or a
functional equivalent thereof. In some embodiments the HDACs is
HDAC1 or HDAC6 a fragment or complex thereof or a functional
equivalent thereof.
[0011] In some embodiments, the lipid kinase/protein kinase is a
PI3K kinase or a fragment thereof or a complex thereof or a
functional equivalent thereof. In some embodiments the PI3K kinase
or a fragment thereof or a complex thereof or a functional
equivalent thereof, is a class I PI3K or a fragment thereof or a
complex thereof or a functional equivalent thereof.
[0012] In some embodiments, the protein kinase is a
serine/threonine protein kinase or a lipid kinase or a fragment or
a complex thereof or a functional equivalent thereof. In some
embodiments the serine/threonine protein kinase or a fragment or
complex thereof is an mTOR protein kinase or a fragment thereof, or
a complex thereof or a functional equivalent thereof. In some
embodiments the serine/threonine protein kinase is mTORC1 or a
fragment or complex thereof or a functional equivalent thereof.
[0013] Advantageously, the compound may contain a zinc-binding
group (hydroxamic acid) to inhibit histone deacetylase and other
deacetylase activities as well as a fused pyrimidine core decorated
with substitutes to modulate PI3K-AKT-mTOR pathway. Advantageously,
the compound may contain both a zinc-binding group and a fused
pyrimidine core so that it may simultaneously inhibit histone
deacetylase and other deacetylase activity and modulate the
PI3K-AKT-mTOR pathway. Further advantageously, since the compound
contains a zinc-binding group, it may inhibit any enzyme containing
zinc in its active site. More advantageously, the compound may be
used as an inhibitor of histone deacetylase and other deacetylase
activities. The compound may be used as a modulator for
PI3K-AKT-mTOR pathway.
[0014] More advantageously, the components for each of the target;
that is, for HDAC or kinase, are within the same molecule,
rendering them compatible with each other. This overcomes the issue
of adverse drug-drug interactions. Further, each of the components
for each of the target may be separately optimized in terms of
position at which they are incorporated and substituents which they
may have. This may in turn result in the modulation of the activity
and physico-chemical properties of the drug. Further
advantageously, the compound may work in an additive or synergistic
manner by targeting two separate targets. It is therefore possible
to put the most potent groups for each of the targets within the
same molecule. In addition, the optimization for compatibility of
physico-chemical properties, structure-activity relationships and
testing of the efficacy of the compound may be done on a single
agent without having to test for multiple agents.
[0015] Further advantageously, the compound is designed and created
in such a way that each molecule has a unique potency profile
against each target (ranging from low to high). This suggests that
the compounds may be tweaked so that it can be tailored to have
different efficacy towards different targets for a variety of
indications or applications. For example, the combination of
potency for HDAC/PI3K inhibition can be described to be: high/high,
high/medium, high/low, medium/high, medium/medium, medium/low,
low/high, low/medium and low/low. Depending on the potency
combination, any combination of target enzyme may be targeted.
Further, by having a wide range of compounds that have a variety of
potency combinations, it may be possible to mimic a combinatorial
library for combination therapy. Further advantageously, for a
specific cancer or condition, best compounds may be selected by
evaluating it in vitro and/or in vivo.
[0016] More advantageously, the compounds are small. It has been
found that these small size molecules are less toxic and have less
occurrences of adverse drug effects while maintaining a high level
of activity.
[0017] In practice, design and synthesis of a working multi-target
molecule by hybridising or merging or de novo design is not a
simple task to achieve.
[0018] In some embodiments, the method exposing the one or more
protein kinase(s) to the compound includes administering the
compound to a mammal containing the one or more protein
kinase(s).
[0019] In a further embodiments, there is provided a method of
treating or preventing a condition in a mammal in which inhibition
of one or more protein kinase(s) selected from the group consisting
of a serine/threonine protein kinase or a fragment or a complex
thereof or a functional equivalent thereof and a PI3K kinase or a
fragment or a complex thereof or a functional equivalent thereof,
prevents, inhibits or ameliorates a pathology or a symptomology of
the condition, the method including administration of a
therapeutically effective amount of a compound as defined
above.
[0020] In some embodiments, the conditions are cancer, angiogenic
disorder or pathological angiogenesis, fibrosis, inflammatory
conditions, asthma, neurological disorders, neurodegenerative
disorders, muscle degenerative disorders, autoimmune disorders,
disorders of the blood or disorders of the bone marrow. In some
embodiments the cancer is selected from the group consisting of
hematologic cancer and solid tumor such as myeloproliferative
disorders (idiopathic myelofibrosis, polycythemia vera, essential
thrombocythemia, chronic myeloid leukemia), myeloid metaplasia,
chronic myelomonocytic leukemia, acute lymphocytic leukemia, acute
erythroblastic leukemia, Hodgkin's and Non Hodgkin's disease,
B-cell lymphoma, diffuse large B cell lymphoma, acute T-cell
leukemia, myelodysplastic syndromes, plasma cell disorder, hairy
cell leukemia, kaposi's sarcoma, lymphoma; gynaecologic cancer such
as breast carcinoma, ovarian cancer, cervical cancer, vaginal and
vulva cancer, endometrial hyperplasia; gastrointestinal tract
cancer such as colorectal carcinoma, polyps, liver cancer, gastric
cancer, pancreatic cancer, gall bladder cancer; urinary tract
cancer such as prostate cancer, kidney and renal cancer; urinary
bladder cancer, urethral cancer, penile cancer; skin cancer such as
melanoma; brain tumour such as glioblastoma, neuroblastoma,
astrocytoma, ependynoma, brain-stem gliomas, medulloblastoma,
menigiomas, astrocytoma, oligodendroglioma; head and neck cancer
such as nasopharyngeal carcinoma, laryngeal carcinoma; respiratory
tract cancer such as lung carcinoma (NSCLC and SCLC), mesothelioma;
eye disease such as retinoblastoma; musculo-skeleton diseases such
as osteosarcoma, musculoskeleletal neoplasm; Squamous cell
carcinoma and fibroid tumour.
[0021] In further embodiments, there is provided a use of the
compound as defined above to inhibit one or more deacetylase(s)
selected from the group consisting of HDACs and non-histone
deacetylase or a fragment or a complex thereof or a functional
equivalent thereof.
[0022] In even further embodiments, there is provided a use of the
compound as defined above to inhibit one or more protein kinase(s)
selected from the group consisting of a serine/threonine protein
kinase or a fragment or a complex thereof or a functional
equivalent thereof and a Akt or mTOR kinase or a fragment or a
complex thereof or a functional equivalent thereof.
[0023] In some embodiments, the protein kinase is a PI3K kinase or
a fragment thereof or a complex thereof or a functional equivalent
thereof. In some embodiments the PI3K kinase or a fragment thereof
or a complex thereof or a functional equivalent thereof, is a class
I PI3K or a fragment thereof or a complex thereof or a functional
equivalent thereof.
[0024] In a third aspect, there is provided a method of inhibiting
HDAC and/or PI3K in a cell comprising administering to a cell a
compound as defined above, or a pharmaceutically acceptable form or
prodrug thereof.
[0025] In some embodiments, there is provided a method of
prevention or treatment of a proliferative condition in a subject,
the method including administration of a therapeutically effective
amount of a compound as defined above.
[0026] In a fourth aspect, there is provided a method of treating a
HDAC- or PI3K-related disorder comprising administering to a
subject in need of treatment a compound as defined above, or a
pharmaceutically acceptable form or prodrug thereof, or a
composition as defined above.
[0027] In a fifth aspect, there is provided a method of treating a
HDAC- and PI3K-related disorder comprising administering to a
subject in need of treatment a compound as defined above, or a
pharmaceutically acceptable form or prodrug thereof, or a
composition as defined above.
[0028] In an sixth aspect, there is provided a method of modulating
the self-renewal or differentiation of stem-cells comprising
administering to a subject in need of treatment a compound as
defined above, or a pharmaceutically acceptable form or prodrug
thereof, or a composition as defined above.
[0029] In further embodiments, there is provided a use of the
compound as defined above in the preparation of a medicament for
treating a condition in an animal in which inhibition one or more
deacetylase(s) selected from the group consisting of HDACs and
non-histone deacetylase or a fragment or a complex thereof or a
functional equivalent thereof, prevents, inhibits or ameliorates a
pathology or a symptomology of the condition.
[0030] In further embodiments there is provided a use of the
compound as defined above in the preparation of a medicament for
treating a condition in an animal in which inhibition of one or
more protein kinase(s) selected from the group consisting of a
serine/threonine protein kinase or a fragment or a complex thereof
or a functional equivalent thereof and a PI3K kinase or a fragment
or a complex thereof or a functional equivalent thereof, prevents,
inhibits or ameliorates a pathology or a symptomology of the
condition.
[0031] In a seventh aspect, there is provided a use of the compound
as defined above, or a pharmaceutically acceptable form or prodrug
thereof, or the composition as defined above, in the manufacture of
a medicament for treatment of a HDAC- or PI3K-related disorder.
[0032] In an eighth aspect, there is provided a use of the compound
as defined above, or a pharmaceutically acceptable form or prodrug
thereof, or the composition as defined above in the manufacture of
a medicament for treatment of a HDAC- and PI3K-related
disorder.
[0033] In a ninth aspect, there is provided a use of the compound
as defined above, or a pharmaceutically acceptable form or prodrug
thereof, or the composition as defined above, in the manufacture of
a medicament for modulating the self-renewal or differentiation of
stem-cells.
[0034] In further embodiments, there is provided a use of the
compound as defined above or a pharmaceutically acceptable salt,
N-oxide or prodrug thereof in the treatment of a condition in which
inhibition of one or more protein kinase(s) selected from the group
consisting of a serine/threonine protein kinase or a fragment or a
complex thereof or a functional equivalent thereof and a PI3K
kinase or a fragment or a complex thereof or a functional
equivalent thereof, prevents, inhibits or ameliorates a pathology
or a symptomology of the condition.
[0035] In some embodiments the protein kinase is a serine/threonine
protein kinase or a fragment or a complex thereof or a functional
equivalent thereof. In some embodiments the serine/threonine
protein kinase or a fragment or complex thereof is an mTOR protein
kinase or a fragment thereof, or a complex thereof or a functional
equivalent thereof. In some embodiments the serine/threonine
protein kinase is mTORC1 or a fragment or complex thereof or a
functional equivalent thereof.
[0036] In some embodiments, the protein kinase is a PI3K kinase or
a fragment thereof or a complex thereof or a functional equivalent
thereof. In some embodiments the PI3K kinase or a fragment thereof
or a complex thereof or a functional equivalent thereof, is a class
I PI3K or a fragment thereof or a complex thereof or a functional
equivalent thereof.
[0037] In some embodiments, the serine/threonine protein kinase or
a fragment or complex thereof is an Akt protein kinase or a
fragment thereof, or a complex thereof or a functional equivalent
thereof.
[0038] In further embodiments, there is provided a method of
reprogramming cells to induced pluripotent stem cells (iPS cells).
The method comprises administration of a therapeutically effective
amount of a compound as defined above to cells isolated from a
subject.
[0039] In further embodiments, there is provided a use of the
compound as defined above in the preparation of a medicament for
treating a proliferative condition in a subject.
[0040] In some embodiments the conditions are cancer, angiogenic
disorder or pathological angiogenesis, fibrosis, inflammatory
conditions, asthma, neurological disorders, neurodegenerative
disorders, muscle degenerative disorders, autoimmune disorders,
disorders of the blood or disorders of the bone marrow. In some
embodiments the cancer is selected from the group consisting of
hematologic cancer and solid tumor such as myeloproliferative
disorders (idiopathic myelofibrosis, polycythemia vera, essential
thrombocythemia, chronic myeloid leukemia), myeloid metaplasia,
chronic myelomonocytic leukemia, acute lymphocytic leukemia, acute
erythroblastic leukemia, Hodgkin's and Non Hodgkin's disease,
B-cell lymphoma, diffuse large B cell lymphoma, acute T-cell
leukemia, myelodysplastic syndromes, plasma cell disorder, hairy
cell leukemia, kaposi's sarcoma, lymphoma; gynaecologic cancer such
as breast carcinoma, ovarian cancer, cervical cancer, vaginal and
vulva cancer, endometrial hyperplasia; gastrointestinal tract
cancer such as colorectal carcinoma, polyps, liver cancer, gastric
cancer, pancreatic cancer, gall bladder cancer; urinary tract
cancer such as prostate cancer, kidney and renal cancer; urinary
bladder cancer, urethral cancer, penile cancer; skin cancer such as
melanoma; brain tumour such as glioblastoma, neuroblastoma,
astrocytoma, ependynoma, brain-stem gliomas, medulloblastoma,
menigiomas, astrocytoma, oligodendroglioma; head and neck cancer
such as nasopharyngeal carcinoma, laryngeal carcinoma; respiratory
tract cancer such as lung carcinoma (NSCLC and SCLC), mesothelioma;
eye disease such as retinoblastoma; musculo-skeleton diseases such
as osteosarcoma, musculoskeleletal neoplasm; Squamous cell
carcinoma and fibroid tumour.
[0041] Advantageously, the compounds as defined above demonstrated
inhibitory activities against HDAC enzymes and PI3K kinases and
anti-proliferative activities against a variety of human tumour
cell lines. Most of the compound as defined above demonstrated good
drug-like properties, that is, in vitro metabolic stability,
solubility and desirable lipophilicity. Further advantageously, the
compounds also showed activity against multi-targets in tumor
cells, i.e., hyperacetylation of histones and .alpha.-tubulin due
to inhibition of HDACs; PI3K-AKT-mTOR pathway: reduction of
phosphor-Akt (Ser473) or inhibition the activity of mTORC2, and
reduction of phospho-P70S6K (Thr389)/phospho-P85S6K (Thr412),
phospho-S6 (Ser240/244) and phospho-4E-BP1 (Thr37/46) or inhibition
of the activity of mTOCR1. More advantageously, these compounds
also induced cell apoptosis in PC-3 cells and MV-4-11 cells, cell
death in MV-4-11 cells, much more efficiently than PI3k inhibitor
GDC-0941.
[0042] Advantageously, these compounds also modulated biological
drug targets in tumor models. The compounds induced histone
hyperacetylation in PC-3 prostate tumors when orally dosed in
tumor-bearing mice and induced histone hyperacetylation in MV4-11
xenograft tumors via different routes of administration. The
compound also demonstrated excellent antitumor activity in HCC
models such as NCr nude mice HepG2 xenograft model and CB17 scid
mice HepG2 xenograft model as well as HuH-7 HCC xenograft model.
The compound was also demonstrated to have broad antitumor activity
in a number of xenograft models when dosed orally in 4T1 mouse
metastatic breast cancer model, NCI-H460 lung cancer xenograft
model and MV4-11 leukaemia xenograft model.
[0043] The disclosure further relates to a process for synthesizing
the compound of formula (I) and its precursors.
[0044] In a tenth aspect, there is provided a process for
synthesizing the compound of formula (I) comprising the steps of;
(a) providing a halogen-disubstituted purine-based or halogen
di-substituted fused pyrimidine-based compound; (b) alkylating the
amine (--NH-- group) in the compound of step (a); (c) selectively
or sequentially displacing the halide atoms of the intermediary
compound of step (b) with an optionally substituted boronic ester
or an optionally substituted amine to form a substituted aromatic
or a substituted amine, respectively; (d) selectively coupling the
intermediary compound of step (c) with a protected hydroxamic acid
group having the structure
-L.sup.1-R.sup.5-L.sup.2-R.sup.6-L.sup.3-CON(R.sup.a)OR.sup.b or an
ester (hydroxamic acid precursor); and (e) converting the protected
hydroxamate or the ester of the intermediary compound of step (d)
to a hydroxamic acid under reaction conditions to form the compound
of formula (I).
[0045] In a eleventh aspect, there is provided a process for
synthesizing the compound of formula (I), comprising the steps of;
(a) providing a halogen-disubstituted purine-based or halogen
di-substituted fused pyrimidine-based compound; (b) selectively
displacing one of the halide atoms of said compound with an
optionally substituted boronic ester or an optionally substituted
amine to form a substituted aromatic or a substituted amine,
respectively; (c) alkylating the amine (--NH-- group) in the
intermediary compound of step (b); (d) selectively displacing the
remaining halide atom of the intermediary compound of step (c) with
an optionally substituted boronic ester or an optionally
substituted amine to form a substituted aromatic or a substituted
amine, respectively; (e) selectively coupling the intermediary
compound of step (d) with a protected hydroxamic acid group having
the structure
-L.sup.1-R.sup.5-L.sup.2-R.sup.6-L.sup.3-CON(R.sup.a)OR.sup.b or an
ester (hydroxamic acid precursor); and (f) converting the protected
hydroxamate or the ester of the intermediary compound of step (e)
to a hydroxamic acid under reaction conditions to form the compound
of formula (I).
[0046] In a twelfth aspect, there is provided a process for
synthesizing the compound of formula (I);
##STR00002##
comprising the steps of; (a) providing a halogen-disubstituted
purine-based or halogen di-substituted fused pyrimidine-based
compound; (b) alkylating the amine in the compound of step (a); (c)
selectively or sequentially displacing the halide atoms of the
intermediary compound of step (b) with an optionally substituted
boronic ester or an optionally substituted amine to form a
substituted aromatic or a substituted amine, respectively; (d)
alkylating, in the intermediary compound of step (c), the carbon
atom that corresponds to the Y-position of formula (I); (e)
selectively coupling the intermediary compound of step (d) with a
protected hydroxamic acid group having the structure
-L.sup.1R.sup.5-L.sup.2-R.sup.6-L.sup.3-CON(R.sup.a)OR.sup.b or an
ester (hydroxamic ester precursor); and (f) converting the
protected hydroxamate or the ester of the intermediary compound of
step (e) to a hydroxamic acid under reaction conditions to form the
compound of formula (I).
[0047] In a thirteenth aspect, there is provided a process for
synthesizing the compound of formula (I), comprising the steps of;
(a) providing a halogen-disubstituted purine-based or halogen
di-substituted fused pyrimidine-based compound; (b) selectively
displacing one of the halide atoms of said compound with an
optionally substituted boronic ester or an optionally substituted
amine to form a substituted aromatic or a substituted amine,
respectively; (c) alkylating the amine (--NH-- group) in the
intermediary compound of step (b); (d) alkylating, in the
intermediary compound of step (c), the carbon atom that corresponds
to the Y-position of formula (I); (e) selectively displacing the
remaining halide atom of the intermediary compound of step (d) with
an optionally substituted boronic ester or an optionally
substituted amine to form a substituted aromatic or a substituted
amine, respectively; (f) selectively coupling the compound of step
(e) with a protected hydroxamic acid group having the structure
-L.sup.1-R.sup.5-L.sup.2-R.sup.6-L.sup.3-CON(R.sup.a)OR.sup.b or an
ester (hydroxamic acid precursor); and (g) converting the protected
hydroxamate or the ester of the intermediary compound of step (f)
to a hydroxamic acid under reaction conditions to form the compound
of formula (I).
[0048] These and other features of the present teachings are set
forth herein.
Definitions
[0049] In this specification a number of terms are used which are
well known to a skilled addressee. Nevertheless for the purposes of
clarity a number of terms will be defined. The following words and
terms used herein shall have the meaning indicated:
[0050] In the definitions of a number of substituents below it is
stated that "the group may be a terminal group or a bridging
group". This is intended to signify that the use of the term is
intended to encompass the situation where the group is a linker
between two other portions of the molecule as well as where it is a
terminal moiety. Using the term alkyl as an example, some
publications would use the term "alkylene" for a bridging group and
hence in these other publications there is a distinction between
the terms "alkyl" (terminal group) and "alkylene" (bridging group).
In the present application no such distinction is made and most
groups may be either a bridging group or a terminal group.
[0051] "Acyl" means an R--C(.dbd.O)-- group in which the R group
may be an optionally substituted alkyl, optionally substituted
cycloalkyl, optionally substituted heterocycloalkyl, optionally
substituted aryl or optionally substituted heteroaryl group as
defined herein. Examples of acyl include acetyl, benzoyl and amino
acid derived aminoacyl. The group may be a terminal group or a
bridging group. If the group is a terminal group it is bonded to
the remainder of the molecule through the carbonyl carbon.
[0052] "Acylamino" means an R--C(.dbd.O)--NH-- group in which the R
group may be an alkyl, cycloalkyl, heterocycloalkyl; aryl or
heteroaryl group as defined herein. The group may be a terminal
group or a bridging group. If the group is a terminal group it is
bonded to the remainder of the molecule through the nitrogen
atom.
[0053] "Alkenyl" as a group or part of a group denotes an aliphatic
hydrocarbon group containing at least one carbon-carbon double bond
and which may be straight or branched preferably having 2-12 carbon
atoms, more preferably 2-10 carbon atoms, most preferably 2-6
carbon atoms, in the normal chain. The group may contain a
plurality of double bonds in the normal chain and the orientation
about each is independently E or Z. Exemplary alkenyl groups
include, but are not limited to, ethenyl, propenyl, butenyl,
pentenyl, hexenyl, heptenyl, octenyl and nonenyl. The group may be
a terminal group or a bridging group.
[0054] "Alkenyloxy" refers to an alkenyl-O-- group in which alkenyl
is as defined herein. Preferred alkenyloxy groups are
C.sub.1-C.sub.6 alkenyloxy groups. The group may be a terminal
group or a bridging group. If the group is a terminal group it is
bonded to the remainder of the molecule through the oxygen
atom.
[0055] "Alkyl" as a group or part of a group refers to a straight
or branched aliphatic hydrocarbon group, preferably a
C.sub.1-C.sub.12 alkyl, more preferably a C.sub.1-C.sub.10 alkyl,
most preferably C.sub.1-C.sub.6 unless otherwise noted. Examples of
suitable straight and branched C.sub.1-C.sub.6 alkyl substituents
include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl,
t-butyl, hexyl, and the like. The group may be a terminal group or
a bridging group.
[0056] "Alkylamino" includes both mono-alkylamino and dialkylamino,
unless specified. "Mono-alkylamino" means a Alkyl-NH-- group, in
which alkyl is as defined herein. "Dialkylamino" means a
(alkyl).sub.2N-group, in which each alkyl may be the same or
different and are each as defined herein for alkyl. The alkyl group
is preferably a C.sub.1-C.sub.6 alkyl group. The group may be a
terminal group or a bridging group. If the group is a terminal
group it is bonded to the remainder of the molecule through the
nitrogen atom.
[0057] "Alkylaminocarbonyl" refers to a group of the formula
(Alkyl).sub.x(H).sub.yNC(.dbd.O)-- in which alkyl is as defined
herein, x is 1 or 2, and the sum of X+Y=2. The group may be a
terminal group or a bridging group. If the group is a terminal
group it is bonded to the remainder of the molecule through the
carbonyl carbon.
[0058] "Alkyloxy" refers to an alkyl-O-- group in which alkyl is as
defined herein. Preferably the alkyloxy is a
C.sub.1-C.sub.6alkyloxy. Examples include, but are not limited to,
methoxy and ethoxy. The group may be a terminal group or a bridging
group.
[0059] "Alkyloxyalkyl" refers to an alkyloxy-alkyl-group in which
the alkyloxy and alkyl moieties are as defined herein. The group
may be a terminal group or a bridging group. If the group is a
terminal group it is bonded to the remainder of the molecule
through the alkyl group.
[0060] "Alkyloxyary" refers to an alkyloxy-aryl-group in which the
alkyloxy and aryl moieties are as defined herein. The group may be
a terminal group or a bridging group. If the group is a terminal
group it is bonded to the remainder of the molecule through the
aryl group.
[0061] "Alkyloxycarbonyl" refers to an alkyl-O--C(.dbd.O)-- group
in which alkyl is as defined herein. The alkyl group is preferably
a C.sub.1-C.sub.6 alkyl group. Examples include, but are not
limited to, methoxycarbonyl and ethoxycarbonyl. The group may be a
terminal group or a bridging group. If the group is a terminal
group it is bonded to the remainder of the molecule through the
carbonyl carbon.
[0062] "Alkyloxycycloalkyl" refers to an alkyloxy-cycloalkyl-group
in which the alkyloxy and cycloalkyl moieties are as defined
herein. The group may be a terminal group or a bridging group. If
the group is a terminal group it is bonded to the remainder of the
molecule through the cycloalkyl group.
[0063] "Alkyloxyheteroaryl" refers to an alkyloxy-heteroaryl-group
in which the alkyloxy and heteroaryl moieties are as defined
herein. The group may be a terminal group or a bridging group. If
the group is a terminal group it is bonded to the remainder of the
molecule through the heteroaryl group.
[0064] "Alkyloxyheterocycloalkyl" refers to an
alkyloxy-heterocycloalkyl-group in which the alkyloxy and
heterocycloalkyl moieties are as defined herein. The group may be a
terminal group or a bridging group. If the group is a terminal
group it is bonded to the remainder of the molecule through the
heterocycloalkyl group.
[0065] "Alkylsulfinyl" means an alkyl-S--(.dbd.O)-- group in which
alkyl is as defined herein. The alkyl group is preferably a
C.sub.1-C.sub.6 alkyl group. Exemplary alkylsulfinyl groups
include, but not limited to, methylsulfinyl and ethylsulfinyl. The
group may be a terminal group or a bridging group. If the group is
a terminal group it is bonded to the remainder of the molecule
through the sulfur atom.
[0066] "Alkylsulfonyl" refers to an alkyl-S(.dbd.O).sub.2-- group
in which alkyl is as defined above. The alkyl group is preferably a
C.sub.1-C.sub.6 alkyl group. Examples include, but not limited to
methylsulfonyl and ethylsulfonyl. The group may be a terminal group
or a bridging group. If the group is a terminal group it is bonded
to the remainder of the molecule through the sulfur atom.
[0067] "Alkynyl" as a group or part of a group means an aliphatic
hydrocarbon group containing a carbon-carbon triple bond and which
may be straight or branched preferably having from 2-12 carbon
atoms, more preferably 2-10 carbon atoms, more preferably 2-6
carbon atoms in the normal chain. Exemplary structures include, but
are not limited to, ethynyl and propynyl. The group may be a
terminal group or a bridging group.
[0068] "Alkynyloxy" refers to an alkynyl-O-- group in which alkynyl
is as defined herein. Preferred alkynyloxy groups are
C.sub.1-C.sub.6 alkynyloxy groups. The group may be a terminal
group or a bridging group. If the group is a terminal group it is
bonded to the remainder of the molecule through the oxygen
atom.
[0069] "Amino acid" as a group or part of a group means having at
least one primary, secondary, tertiary or quaternary amino group,
and at least one acid group, wherein the acid group may be a
carboxylic, sulfonic, or phosphonic acid, or mixtures thereof. The
amino groups may be "alpha", "beta", "gamma" . . . to "omega" with
respect to the acid group(s). The amino acid may be natural or
synthetic, and may include their derivatives. The backbone of the
"amino acid" may be substituted with one or more groups selected
from halogen, hydroxy, guanido, heterocyclic groups. Thus the term
"amino acids" also includes within its scope glycine, alanine,
valine, leucine, isoleucine, methionine, proline, phenylalanine,
tryptophane, serine, threonine, cysteine, tyrosine, asparagine,
glutamine, asparte, glutamine, lysine, arginine and histidine,
taurine, betaine, N-methylalanine etc. (L) and (D) forms of amino
acids are included in the scope of this disclosure. Additionally,
the amino acids suitable for use in the present disclosure may be
derivatized to include amino acids that are hydroxylated,
phosphorylated, sulfonated, acylated, and glycosylated, to name a
few.
[0070] "Amino acid residue" refers to amino acid structures that
lack a hydrogen atom of the amino group (--NH--CHR--COOH), or the
hydroxy moiety of the carboxygroup (NH2-CHR--CO--), or both
(--NH--CHR--CO--).
[0071] "Amino" refers to groups of the form --NR.sub.aR.sub.b
wherein R.sub.a and R.sub.b are individually selected from the
group including but not limited to hydrogen, optionally substituted
alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, and optionally substituted aryl groups.
[0072] "Aminoalkyl" means an NH.sub.2-alkyl-group in which the
alkyl group is as defined herein. The group may be a terminal group
or a bridging group. If the group is a terminal group it is bonded
to the remainder of the molecule through the alkyl group.
[0073] "Aminosulfonyl" means an NH.sub.2--S(.dbd.O).sub.2-- group.
The group may be a terminal group or a bridging group. If the group
is a terminal group it is bonded to the remainder of the molecule
through the sulfur atom.
[0074] "Aryl" as a group or part of a group denotes (i) an
optionally substituted monocyclic, or fused polycyclic, aromatic
carbocycle (ring structure having ring atoms that are all carbon)
preferably having from 5 to 12 atoms per ring. Examples of aryl
groups include phenyl, naphthyl, and the like; (ii) an optionally
substituted partially saturated bicyclic aromatic carbocyclic
moiety in which a phenyl and a C.sub.5-7 cycloalkyl or C.sub.5-7
cycloalkenyl group are fused together to form a cyclic structure,
such as tetrahydronaphthyl, indenyl or indanyl. The group may be a
terminal group or a bridging group. Typically an aryl group is a
C.sub.6-C.sub.18 aryl group.
[0075] "Arylalkenyl" means an aryl-alkenyl-group in which the aryl
and alkenyl are as defined herein. Exemplary arylalkenyl groups
include phenylallyl. The group may be a terminal group or a
bridging group. If the group is a terminal group it is bonded to
the remainder of the molecule through the alkenyl group.
[0076] "Arylalkyl" means an aryl-alkyl-group in which the aryl and
alkyl moieties are as defined herein. Preferred arylalkyl groups
contain a C.sub.1-5 alkyl moiety. Exemplary arylalkyl groups
include benzyl, phenethyl, 1-naphthalenemethyl and
2-naphthalenemethyl. The group may be a terminal group or a
bridging group. If the group is a terminal group it is bonded to
the remainder of the molecule through the alkyl group.
[0077] "Arylalkyloxy" refers to an aryl-alkyl-O-- group in which
the alkyl and aryl are as defined herein. The group may be a
terminal group or a bridging group. If the group is a terminal
group it is bonded to the remainder of the molecule through the
oxygen atom.
[0078] "Arylamino" includes both mono-arylamino and di-arylamino
unless specified. Mono-arylamino means a group of formula arylNH--,
in which aryl is as defined herein. di-arylamino means a group of
formula (aryl).sub.2N-where each aryl may be the same or different
and are each as defined herein for aryl. The group may be a
terminal group or a bridging group. If the group is a terminal
group it is bonded to the remainder of the molecule through the
nitrogen atom.
[0079] "Arylheteroalkyl" means an aryl-heteroalkyl-group in which
the aryl and heteroalkyl moieties are as defined herein. The group
may be a terminal group or a bridging group. If the group is a
terminal group it is bonded to the remainder of the molecule
through the heteroalkyl group.
[0080] "Aryloxy" refers to an aryl-O-- group in which the aryl is
as defined herein. Preferably the aryloxy is a
C.sub.6-C.sub.18aryloxy, more preferably a C.sub.6-C.sub.10aryloxy.
The group may be a terminal group or a bridging group. If the group
is a terminal group it is bonded to the remainder of the molecule
through the oxygen atom.
[0081] "Arylsulfonyl" means an aryl-S(.dbd.O).sub.2-- group in
which the aryl group is as defined herein. The group may be a
terminal group or a bridging group. If the group is a terminal
group it is bonded to the remainder of the molecule through the
sulfur atom.
[0082] A "bond" is a linkage between atoms in a compound or
molecule. The bond may be a single bond, a double bond, or a triple
bond.
[0083] "Cycloalkenyl" means a non-aromatic monocyclic or
multicyclic ring system containing at least one carbon-carbon
double bond and preferably having from 5-10 carbon atoms per ring.
Exemplary monocyclic cycloalkenyl rings include cyclopentenyl,
cyclohexenyl or cycloheptenyl. The cycloalkenyl group may be
substituted by one or more substituent groups. A cycloalkenyl group
typically is a C.sub.3-C.sub.12 alkenyl group. The group may be a
terminal group or a bridging group.
[0084] "Cycloalkyl" refers to a saturated monocyclic or fused or
spiro polycyclic, carbocycle preferably containing from 3 to 9
carbons per ring, such as cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl and the like, unless otherwise specified. It includes
monocyclic systems such as cyclopropyl and cyclohexyl, bicyclic
systems such as decalin, and polycyclic systems such as adamantane.
A cycloalkyl group typically is a C.sub.3-C.sub.12 alkyl group. The
group may be a terminal group or a bridging group.
[0085] "Cycloalkylalkyl" means a cycloalkyl-alkyl-group in which
the cycloalkyl and alkyl moieties are as defined herein. Exemplary
monocycloalkylalkyl groups include cyclopropylmethyl,
cyclopentylmethyl, cyclohexylmethyl and cycloheptylmethyl. The
group may be a terminal group or a bridging group. If the group is
a terminal group it is bonded to the remainder of the molecule
through the alkyl group.
[0086] "Cycloalkylalkenyl" means a cycloalkyl-alkenyl-group in
which the cycloalkyl and alkenyl moieties are as defined herein.
The group may be a terminal group or a bridging group. If the group
is a terminal group it is bonded to the remainder of the molecule
through the alkenyl group.
[0087] "Cycloalkylheteroalkyl" means a cycloalkyl-heteroalkyl-group
in which the cycloalkyl and heteroalkyl moieties are as defined
herein. The group may be a terminal group or a bridging group. If
the group is a terminal group it is bonded to the remainder of the
molecule through the heteroalkyl group.
[0088] "Cycloalkyloxy" refers to a cycloalkyl-O-- group in which
cycloalkyl is as defined herein. Preferably the cycloalkyloxy is a
C.sub.1-C.sub.6cycloalkyloxy. Examples include, but are not limited
to, cyclopropanoxy and cyclobutanoxy. The group may be a terminal
group or a bridging group. If the group is a terminal group it is
bonded to the remainder of the molecule through the oxygen
atom.
[0089] "Cycloalkenyloxy" refers to a cycloalkenyl-O-- group in
which the cycloalkenyl is as defined herein. Preferably the
cycloalkenyloxy is a C.sub.1-C.sub.6cycloalkenyloxy. The group may
be a terminal group or a bridging group. If the group is a terminal
group it is bonded to the remainder of the molecule through the
oxygen atom.
[0090] "Cycloamino" refers to a saturated monocyclic, bicyclic, or
polycyclic ring containing at least one nitrogen in at least one
ring. Each ring is preferably from 3 to 10 membered, more
preferably 4 to 7 membered. The group may be a terminal group or a
bridging group. If the group is a terminal group it is bonded to
the remainder of the molecule through the nitrogen atom.
[0091] "Haloalkyl" refers to an alkyl group as defined herein in
which one or more of the hydrogen atoms has been replaced with a
halogen atom selected from the group consisting of fluorine,
chlorine, bromine and iodine. A haloalkyl group typically has the
formula C.sub.nH.sub.(2n+1-m)X.sub.m wherein each X is
independently selected from the group consisting of F, Cl, Br and
I. In groups of this type n is typically from 1 to 10, more
preferably from 1 to 6, most preferably 1 to 3. m is typically 1 to
6, more preferably 1 to 3. Examples of haloalkyl include
fluoromethyl, difluoromethyl and trifluoromethyl.
[0092] "Haloalkenyl" refers to an alkenyl group as defined herein
in which one or more of the hydrogen atoms has been replaced with a
halogen atom independently selected from the group consisting of F,
Cl, Br and I.
[0093] "Haloalkynyl" refers to an alkynyl group as defined herein
in which one or more of the hydrogen atoms has been replaced with a
halogen atom independently selected from the group consisting of F,
Cl, Br and I.
[0094] "Halogen" represents chlorine, fluorine, bromine or
iodine.
[0095] "Heteroalkyl" refers to a straight- or branched-chain alkyl
group preferably having from 2 to 12 carbons, more preferably 2 to
6 carbons in the chain, one or more of which has been replaced by a
heteroatom selected from S, O, P and N. Exemplary heteroalkyls
include alkyl ethers, secondary and tertiary alkyl amines, amides,
alkyl sulfides, and the like. Examples of heteroalkyl also include
hydroxyC-C.sub.6alkyl, C.sub.1-C.sub.6alkyloxyC.sub.1-C.sub.6alkyl,
aminoC.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkylaminoC.sub.1-C.sub.6alkyl, and
di(C.sub.1-C.sub.6alkyl)aminoC.sub.1-C.sub.6alkyl. The group may be
a terminal group or a bridging group.
[0096] "Heteroalkyloxy" refers to an heteroalkyl-O-- group in which
heteroalkyl is as defined herein. Preferably the heteroalkyloxy is
a C.sub.1-C.sub.6heteroalkyloxy. The group may be a terminal group
or a bridging group.
[0097] "Heteroaryl" either alone or part of a group refers to
groups containing an aromatic ring (preferably a 5 or 6 membered
aromatic ring) having one or more heteroatoms as ring atoms in the
aromatic ring with the remainder of the ring atoms being carbon
atoms. Suitable heteroatoms include nitrogen, oxygen and sulphur.
Examples of heteroaryl include thiophene, benzothiophene,
benzofuran, benzimidazole, benzoxazole, benzothiazole,
benzisothiazole, naphtho[2,3-b]thiophene, furan, isoindolizine,
xantholene, phenoxatine, pyrrole, imidazole, pyrazole, pyridine,
pyrazine, pyrimidine, pyridazine, tetrazole, indole, isoindole,
1H-indazole, purine, quinoline, isoquinoline, phthalazine,
naphthyridine, quinoxaline, cinnoline, carbazole, phenanthridine,
acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole,
isooxazole, furazane, phenoxazine, 2-, 3- or 4-pyridyl, 2-, 3-, 4-,
5-, or 8-quinolyl, 1-, 3-, 4-, or 5-isoquinolinyl 1-, 2-, or
3-indolyl, and 2-, or 3-thienyl. A heteroaryl group is typically a
C.sub.1-C.sub.18 heteroaryl group. A heteroaryl group may comprise
3 to 8 ring atoms. A heteroaryl group may comprise 1 to 3
heteroatoms independently selected from the group consisting of N,
O and S. The group may be a terminal group or a bridging group.
[0098] "Heteroarylalkyl" means a heteroaryl-alkyl group in which
the heteroaryl and alkyl moieties are as defined herein. Preferred
heteroarylalkyl groups contain a lower alkyl moiety. Exemplary
heteroarylalkyl groups include pyridylmethyl. The group may be a
terminal group or a bridging group. If the group is a terminal
group it is bonded to the remainder of the molecule through the
alkyl group.
[0099] "Heteroarylalkenyl" means a heteroaryl-alkenyl-group in
which the heteroaryl and alkenyl moieties are as defined herein.
The group may be a terminal group or a bridging group. If the group
is a terminal group it is bonded to the remainder of the molecule
through the alkenyl group.
[0100] "Heteroarylheteroalkyl" means a heteroaryl-heteroalkyl-group
in which the heteroaryl and heteroalkyl moieties are as defined
herein. The group may be a terminal group or a bridging group. If
the group is a terminal group it is bonded to the remainder of the
molecule through the heteroalkyl group.
[0101] "Heteroarylamino" refers to groups containing an aromatic
ring (preferably 5 or 6 membered aromatic ring) having at least one
nitrogen and at least another heteroatom as ring atoms in the
aromatic ring, preferably from 1 to 3 heteroatoms in at least one
ring. Suitable heteroatoms include nitrogen, oxygen and sulphur.
Arylamino and aryl is as defined herein. The group may be a
terminal group or a bridging group. If the group is a terminal
group it is bonded to the remainder of the molecule through the
nitrogen atom.
[0102] "Heteroaryloxy" refers to a heteroaryl-O-- group in which
the heteroaryl is as defined herein. Preferably the heteroaryloxy
is a C.sub.1-C.sub.18heteroaryloxy. The group may be a terminal
group or a bridging group. If the group is a terminal group it is
bonded to the remainder of the molecule through the oxygen
atom.
[0103] "Heterocyclic" refers to saturated, partially unsaturated or
fully unsaturated monocyclic, bicyclic or polycyclic ring system
containing at least one heteroatom selected from the group
consisting of nitrogen, sulfur and oxygen as a ring atom. Examples
of heterocyclic moieties include heterocycloalkyl,
heterocycloalkenyl and heteroaryl.
[0104] "Heterocycloalkenyl" refers to a heterocycloalkyl as defined
herein but containing at least one double bond. A
heterocycloalkenyl group typically is a C.sub.2-C.sub.12
heterocycloalkenyl group. The group may be a terminal group or a
bridging group.
[0105] "Heterocycloalkyl" refers to a saturated monocyclic,
bicyclic, or polycyclic ring containing at least one heteroatom
selected from nitrogen, sulfur, oxygen, preferably from 1 to 3
heteroatoms in at least one ring. Each ring is preferably from 3 to
10 membered, more preferably 4 to 7 membered. Examples of suitable
heterocycloalkyl substituents include pyrrolidyl, tetrahydrofuryl,
tetrahydrothiofuranyl, piperidyl, piperazyl, tetrahydropyranyl,
morphilino, 1,3-diazapane, 1,4-diazapane, 1,4-oxazepane, and
1,4-oxathiapane. A heterocycloalkyl group typically is a
C.sub.2-C.sub.12 heterocycloalkyl group. A heterocycloalkyl group
may comprise 3 to 8 ring atoms. A heterocycloalkyl group may
comprise 1 to 3 heteroatoms independently selected from the group
consisting of N, O and S. The group may be a terminal group or a
bridging group.
[0106] "Heterocycloalkylalkyl" refers to a
heterocycloalkyl-alkyl-group in which the heterocycloalkyl and
alkyl moieties are as defined herein. Exemplary
heterocycloalkylalkyl groups include (2-tetrahydrofuryl)methyl,
(2-tetrahydrothiofuranyl) methyl. The group may be a terminal group
or a bridging group. If the group is a terminal group it is bonded
to the remainder of the molecule through the alkyl group.
[0107] "Heterocycloalkylalkenyl" refers to a
heterocycloalkyl-alkenyl-group in which the heterocycloalkyl and
alkenyl moieties are as defined herein. The group may be a terminal
group or a bridging group. If the group is a terminal group it is
bonded to the remainder of the molecule through the alkenyl
group.
[0108] "Heterocycloalkylheteroalkyl" means a
heterocycloalkyl-heteroalkyl-group in which the heterocycloalkyl
and heteroalkyl moieties are as defined herein. The group may be a
terminal group or a bridging group. If the group is a terminal
group it is bonded to the remainder of the molecule through the
heteroalkyl group.
[0109] "Heterocycloalkyloxy" refers to a heterocycloalkyl-O-- group
in which the heterocycloalkyl is as defined herein. Preferably the
heterocycloalkyloxy is a C.sub.1-C.sub.6heterocycloalkyloxy. The
group may be a terminal group or a bridging group. If the group is
a terminal group it is bonded to the remainder of the molecule
through the oxygen atom.
[0110] "Heterocycloalkenyloxy" refers to a heterocycloalkenyl-O--
group in which heterocycloalkenyl is as defined herein. Preferably
the Heterocycloalkenyloxy is a C.sub.1-C.sub.6
Heterocycloalkenyloxy. The group may be a terminal group or a
bridging group. If the group is a terminal group it is bonded to
the remainder of the molecule through the oxygen atom.
[0111] "Heterocycloamino" refers to a saturated monocyclic,
bicyclic, or polycyclic ring containing at least one nitrogen and
at least another heteroatom selected from nitrogen, sulfur, oxygen,
preferably from 1 to 3 heteroatoms in at least one ring. Each ring
is preferably from 3 to 10 membered, more preferably 4 to 7
membered. The group may be a terminal group or a bridging group. If
the group is a terminal group it is bonded to the remainder of the
molecule through the nitrogen atom.
[0112] "Hydroxyalkyl" refers to an alkyl group as defined herein in
which one or more of the hydrogen atoms has been replaced with an
OH group. A hydroxyalkyl group typically has the formula
C.sub.nH.sub.(2n+1-x)(OH).sub.x. In groups of this type n is
typically from 1 to 10, more preferably from 1 to 6, most
preferably from 1 to 3. x is typically from 1 to 6, more preferably
from 1 to 4.
[0113] "Lower alkyl" as a group means unless otherwise specified,
an aliphatic hydrocarbon group which may be straight or branched
having 1 to 6 carbon atoms in the chain, more preferably 1 to 4
carbons such as methyl, ethyl, propyl (n-propyl or isopropyl) or
butyl (n-butyl, isobutyl or tertiary-butyl). The group may be a
terminal group or a bridging group.
[0114] "Subject" refers to a human or an animal.
[0115] "Sulfinyl" means an R--S(.dbd.O)-- group in which the R
group may be OH, alkyl, cycloalkyl, heterocycloalkyl; aryl or
heteroaryl group as defined herein. The group may be a terminal
group or a bridging group. If the group is a terminal group it is
bonded to the remainder of the molecule through the sulfur
atom.
[0116] "Sulfinylamino" means an R--S(.dbd.O)--NH-- group in which
the R group may be OH, alkyl, cycloalkyl, heterocycloalkyl; aryl or
heteroaryl group as defined herein. The group may be a terminal
group or a bridging group. If the group is a terminal group it is
bonded to the remainder of the molecule through the nitrogen
atom.
[0117] "Sulfonyl" means an R--S(.dbd.O).sub.2-- group in which the
R group may be OH, alkyl, cycloalkyl, heterocycloalkyl; aryl or
heteroaryl group as defined herein. The group may be a terminal
group or a bridging group. If the group is a terminal group it is
bonded to the remainder of the molecule through the sulfur
atom.
[0118] "Sulfonylamino" means an R--S(.dbd.O).sub.2--NH-- group. The
group may be a terminal group or a bridging group. If the group is
a terminal group it is bonded to the remainder of the molecule
through the nitrogen atom.
[0119] It is understood that included in the family of compounds of
Formula (I) are isomeric forms including diastereoisomers,
enantiomers, tautomers, and geometrical isomers in "E" or "Z"
configurational isomer or a mixture of E and Z isomers. It is also
understood that some isomeric forms such as diastereomers,
enantiomers, and geometrical isomers can be separated by physical
and/or chemical methods and by those skilled in the art.
[0120] Some of the compounds of the disclosed embodiments may exist
as single stereoisomers, racemates, and/or mixtures of enantiomers
and/or diastereomers. All such single stereoisomers, racemates and
mixtures thereof, are intended to be within the scope of the
subject matter described and claimed.
[0121] Additionally, Formula (I) is intended to cover, where
applicable, solvated as well as unsolvated forms of the compounds.
Thus, each formula includes compounds having the indicated
structure, including the hydrated as well as the non-hydrated
forms.
[0122] Further, it is possible that compounds of the invention may
contain more than one asymmetric carbon atom. In those compounds,
the use of a solid line to depict bonds to asymmetric carbon atoms
is meant to indicate that all possible stereoisomers are meant to
be included. The use of a solid line to depict bonds to one or more
asymmetric carbon atoms in a compound of the invention and the use
of a solid or dotted wedge to depict bonds to other asymmetric
carbon atoms in the same compound is meant to indicate that a
mixture of diastereomers is present.
[0123] The term "optionally substituted" as used herein means the
group to which this term refers may be unsubstituted, or may be
substituted with one or more groups independently selected from
alkyl, alkenyl, alkynyl, thioalkyl, cycloalkyl, cycloalkylalkyl,
cycloalkenyl, cycloalkylalkenyl, heterocycloalkyl,
cycloalkylheteroalkyl, cycloalkyloxy, cycloalkenyloxy, cycloamino,
halo, carboxyl, haloalkyl, haloalkynyl, alkynyloxy, heteroalkyl,
heteroalkyloxy, hydroxyl, hydroxyalkyl, alkoxy, thioalkoxy,
alkenyloxy, haloalkoxy, haloalkenyl, haloalkynyl, haloalkenyloxy,
nitro, amino, nitroalkyl, nitroalkenyl, nitroalkynyl,
nitroheterocyclyl, alkylamino, dialkylamino, alkenylamine,
aminoalkyl, alkynylamino, acyl, alkyloxy, alkyloxyalkyl,
alkyloxyaryl, alkyloxycarbonyl, alkyloxycycloalkyl,
alkyloxyheteroaryl, alkyloxyheterocycloalkyl, alkenoyl, alkynoyl,
acylamino, diacylamino, acyloxy, alkylsulfonyloxy, heterocyclic,
heterocycloalkenyl, heterocycloalkyl, heterocycloalkylalkyl,
heterocycloalkylalkenyl, heterocycloalkylheteroalkyl,
heterocycloalkyloxy, heterocycloalkenyloxy, heterocycloxy,
heterocycloamino, haloheterocycloalkyl, alkylsulfinyl,
alkylsulfonyl, alkylsulfenyl, alkylcarbonyloxy, alkylthio,
acylthio, aminosulfonyl, phosphorus-containing groups such as
phosphono and phosphinyl, sulfinyl, sulfinylamino, sulfonyl,
sulfonylamino, aryl, heteroaryl, heteroarylalkyl,
heteroarylalkenyl, heteroarylheteroalkyl, heteroarylamino,
heteroaryloxy, arylalkenyl, arylalkyl, alkylaryl, alkylheteroaryl,
aryloxy, arylsulfonyl, cyano, cyanate, isocyanate, --C(O)NH(alkyl),
and --C(O)N(alkyl).sub.2.
[0124] The term "pharmaceutically acceptable salts" refers to salts
that retain the desired biological activity of the above-identified
compounds, and include pharmaceutically acceptable acid addition
salts and base addition salts. Suitable pharmaceutically acceptable
acid addition salts of compounds of Formula (I) may be prepared
from an inorganic acid or from an organic acid. Examples of such
inorganic acids are hydrochloric, sulfuric, and phosphoric acid.
Appropriate organic acids may be selected from aliphatic,
cycloaliphatic, aromatic, heterocyclic carboxylic and sulfonic
classes of organic acids, examples of which are formic, acetic,
propionic, succinic, glycolic, gluconic, lactic, malic, tartaric,
citric, fumaric, maleic, alkyl sulfonic, arylsulfonic. Additional
information on pharmaceutically acceptable salts can be found in
Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing
Co., Easton, Pa. 1995. In the case of agents that are solids, it is
understood by those skilled in the art that the inventive
compounds, agents and salts may exist in different crystalline or
polymorphic forms, all of which are intended to be within the scope
of the present disclosure and specified formulae.
[0125] "Prodrug" means a compound that undergoes conversion to a
compound of formula (I) within a biological system, usually by
metabolic means (e.g. by hydrolysis, reduction or oxidation). For
example an ester prodrug of a compound of formula (I) containing a
hydroxyl group may be convertible by hydrolysis in vivo to the
parent molecule. Suitable esters of compounds of formula (I)
containing a hydroxyl group, are for example formates, acetates,
citrates, lactates, tartrates, malonates, oxalates, salicylates,
propionates, succinates, fumarates, maleates,
methylene-bis-P3-hydroxynaphthoates, gestisates, isethionates,
di-p-toluoyltartrates, methanesulphonates, ethanesulphonates,
benzenesulphonates, p-toluenesulphonates, cyclohexylsulphamates and
quinates. As another example an ester prodrug of a compound of
formula (I) containing a carboxy group may be convertible by
hydrolysis in vivo to the parent molecule. (Examples of ester
prodrugs are those described by F. J. Leinweber, Drug Metab. Res.,
18:379, 1987). Similarly, an acyl prodrug of a compound of formula
(I) containing an amino group may be convertible by hydrolysis in
vivo to the parent molecule (Many examples of prodrugs for these
and other functional groups, including amines, are described in
Prodrugs: Challenges and Rewards (Parts 1 and 2); Ed V. Stella, R.
Borchardt, M. Hageman, R. Oliyai, H. Maag and J Tilley; Springer,
2007)
[0126] The term "therapeutically effective amount" or "effective
amount" is an amount sufficient to effect beneficial or desired
clinical results. An effective amount can be administered in one or
more administrations. An effective amount is typically sufficient
to palliate, ameliorate, stabilize, reverse, slow or delay the
progression of the disease state.
[0127] The term "functional equivalent" is intended to include
variants of the specific protein kinase species described herein.
It will be understood that kinases may have isoforms, such that
while the primary, secondary, tertiary or quaternary structure of a
given kinase isoform is different to the protoypical kinase, the
molecule maintains biological activity as a protein kinase.
Isoforms may arise from normal allelic variation within a
population and include mutations such as amino acid substitution,
deletion, addition, truncation, or duplication. Also included
within the term "functional equivalent" are variants generated at
the level of transcription. Enzymes (including HDACs and PI3Ks)
have isoforms that arise from transcript variation. Other
functional equivalents include kinases having altered
post-translational modification such as glycosylation.
[0128] The term "reprogramming cells" is intended to include
erasure and remodeling of epigenetic marks, such as DNA
methylation, during mammalian development.
[0129] The word "substantially" does not exclude "completely" e.g.
a composition which is "substantially free" from Y may be
completely free from Y. Where necessary, the word "substantially"
may be omitted from the definition of the invention.
[0130] Unless specified otherwise, the terms "comprising" and
"comprise", and grammatical variants thereof, are intended to
represent "open" or "inclusive" language such that they include
recited elements but also permit inclusion of additional, unrecited
elements.
[0131] As used herein, the term "about", in the context of
concentrations of components of the formulations, typically
means.+-.10% of the stated value, more typically.+-.7.5% of the
stated value, more typically.+-.5% of the stated value, more
typically.+-.4% of the stated value, more typically.+-.3% of the
stated value, more typically, .+-.2% of the stated value, even more
typically.+-.1% of the stated value, and even more
typically.+-.0.5% of the stated value.
[0132] Throughout this disclosure, certain embodiments may be
disclosed in a range format. It should be understood that the
description in range format is merely for convenience and brevity
and should not be construed as an inflexible limitation on the
scope of the disclosed ranges. Accordingly, the description of a
range should be considered to have specifically disclosed all the
possible sub-ranges as well as individual numerical values within
that range. For example, description of a range such as from 1 to 6
should be considered to have specifically disclosed sub-ranges such
as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6,
from 3 to 6 etc., as well as individual numbers within that range,
for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the
breadth of the range.
[0133] Certain embodiments may also be described broadly and
generically herein. Each of the narrower species and subgeneric
groupings falling within the generic disclosure also form part of
the disclosure. This includes the generic description of the
embodiments with a proviso or negative limitation removing any
subject matter from the genus, regardless of whether or not the
excised material is specifically recited herein.
BRIEF DESCRIPTION OF DRAWINGS
[0134] The accompanying drawings illustrate a disclosed embodiment
and serves to explain the principles of the disclosed embodiment.
It is to be understood, however, that the drawings are designed for
purposes of illustration only, and not as a definition of the
limits of the invention.
[0135] FIG. 1 is a scheme showing typical classes of known HDAC
inhibitors and that a typical HDAC inhibitor (vorinostat) comprises
of three parts: zinc-binding group (ZBG), linker, and a lipophilic
and surface recognition "CAP" group. The CAP is sitting outside of
the binding pocket of HDAC enzymes, thus CAP group can be replaced
with a kinase inhibitor scaffold to afford a novel HDAC-kinase dual
inhibitor.
[0136] FIG. 2 shows examples of PI3K/mTOR inhibitors.
[0137] FIG. 3 shows a scheme for a typical procedure to prepare key
intermediate 7 which can be further derivatized to compounds 8, 9,
11 and 12.
[0138] FIG. 4 shows a scheme depicting an alternative displacement
sequence of the two chlorine atoms in compound 1 or 3 and
preparation of compounds 17, 19 and 21.
[0139] FIG. 5 shows a scheme depicting how to further vary R.sup.3
group of compound 7 (scheme 1, FIG. 3) and 18 (scheme 2, FIG. 4)
when R.sup.3 is a hydrogen and X is a nitrogen.
[0140] FIG. 6 shows a scheme depicting an alternative method to
vary R.sup.3 group of compound 14 (scheme 2, FIG. 4) when R.sup.3
is a hydrogen and X is a nitrogen.
[0141] FIG. 7 shows a scheme depicting the general synthetic routes
for further derivatization of readily or commercially available
starting materials (e.g., 4) and procedures for preparation of
protected or substituted hydroxamic acid (38).
[0142] FIG. 8 shows the reaction scheme for the synthesis of
N-hydroxy-4-(3-(9-isopropyl-2-morpholino-9H-purin-6-yl)phenoxy)butanamide-
.
[0143] FIG. 9 shows the reaction scheme for the synthesis of
N-hydroxy-7-(2-(3-(hydroxymethyl)phenyl)-6-morpholino-9H-purin-9-yl)hepta-
namide.
[0144] FIG. 10 shows the reaction scheme for the synthesis of
7-(6-(2-aminopyrimidin-5-yl)-2-morpholino-9H-purin-9-yl)-N-hydroxyheptana-
mide.
[0145] FIG. 11 shows the reaction scheme for the synthesis of
N.sup.1-hydroxy-N.sup.8-(3-(9-isopropyl-2-morpholino-9H-purin-6-yl)phenyl-
)octanediamide.
[0146] FIG. 12 shows the reaction scheme for the synthesis of
(E)-N-hydroxy-3-(4-(((2-(2-(6-methoxypyridin-3-yl)-6-morpholino-9H-purin--
9-yl)ethyl)amino)methyl)phenyl)acrylamide.
[0147] FIG. 13 shows the reaction scheme for the synthesis of
6-((6-(2-aminopyrimidin-5-yl)-9-isopropyl-2-morpholino-9H-purin-8-yl)amin-
o)-N-hydroxyhexanamide.
[0148] FIG. 14 shows the reaction scheme for the synthesis of
4-((((2-(2-aminopyrimidin-5-yl)-9-ethyl-6-morpholino-9H-purin-8-yl)methyl-
)(methyl)amino)methyl)-N-hydroxybenzamide (35 h) and compounds
35a-f.
[0149] FIGS. 15A-D show the inhibition of HDACs and modulation of
PI3k-Akt-mTOR pathway in PC-3 cells treated with the compounds. As
indicated in FIGS. 15A, 15B, 15C and 15D, hyperacetylation of
histone 3 (Lys 9) and .alpha.-tubulin were observed for compound
examples tested and the hyperacetylation effect is dose-dependent
as demonstrated by both EX1 and EX2 (FIG. 15A). The tested examples
also inhibited PI3K-Akt-mTOR pathway with a broad range of
activities. Except EX40 and EX41, they all inhibited
phosphorylation of Akt (Ser473) or activity of mTORC2 (FIG. 15A and
FIG. 15B). FIG. 15C and FIG. 15D demonstrated that they also
inhibited phosphorylation of S6(Ser240/244) or activity of mTORC1
(with exception of EX2, EX40 and EX41). DMSO (0.1%) was used as
blank control for protein phosphorylation and normalized as 100%
when analyzed by ImageJ. PC-3 cells were also treated with insulin
(100 .mu.g/mL) for 30 min and both pAkt and pS6 phosphorylations
were significantly enhanced. The gels were digitized and analyzed
by ImageJ, see FIG. 17 to 20 for details.
[0150] FIG. 16 shows the modulation of the PI3K-AKT-mTOR Pathway in
MCF7 cells treated with the compounds.
[0151] FIGS. 17A-B show the hyperacetylation of histone 3 (Lys 9)
due to inhibition of HDACs in cells treated with the compounds.
FIG. 17A shows PC-3 cells treated with test compounds at above
indicated concentrations (FIG. 15) for 24 h. The graph represents
results from two Western Blot analyses. Vorinostat (10 .mu.M) was
used as positive control, and its acetylation level was normalized
to 100% in ImageJ analysis. FIG. 17B shows MCF7 cells that were
serum starved overnight and treated with test compounds for 2 h
including insulin (20 .mu.g/mL) stimulation in last 30 min for all
samples. Vehicle DMSO (0.1%) was used as blank control, and its
acH3 level was normalized to 1. Y Axis is expressed as Mean.+-.SD
if applicable.
[0152] FIGS. 18A-B show the hyperacetylation of .alpha.-tubulin due
to inhibition of HDAC6 in cells treated with the compounds. FIG.
18A shows PC-3 cells treated with test compounds at above indicated
concentrations (FIG. 15) for 24 h, the graph represents results
from two Western Blot analyses. Vorinostat (10 .mu.M) was used as
positive control, and its acetylation level was normalized to 100%
in ImageJ analysis. FIG. 18B shows MCF7 cells that were serum
starved overnight and treated with test compounds for 2 h including
insulin (20 .mu.g/mL) stimulation in last 30 min for all samples.
Vehicle DMSO (0.1%) as blank control, and its acetylation level was
normalized to 1. Y Axis is expressed as Mean.+-.SD if
applicable.
[0153] FIGS. 19A-B show the modulation of the PI3K-AKT-(mTOR)
Pathway: p-Akt (Ser473) level and activity of mTORC2 in cells
treated with the compounds. FIG. 19A shows PC-3 cells treated with
test compounds at above indicated concentrations (FIG. 15) for 24
h, the graph represents results from two Western Blot analyses.
Vehicle DMSO (0.1%) was used as blank control, and its
phosphorylation level was normalized to 100% in ImageJ analysis.
pAkt level was significantly enhanced in insulin (100 .mu.g/mL, 30
min) treated cells. FIG. 19B shows MCF7 cells serum starved
overnight and treated with test compounds for 2 h including insulin
stimulation (20 .mu.g/mL) in last 30 min for all samples. Vehicle
DMSO (0.1%) was used as blank control, its phosphorylation level
was normalized to 100%. Y Axis is expressed as Mean.+-.SD if
applicable.
[0154] FIGS. 20A-D show the modulation of the PI3K-AKT-(mTOR)
Pathway: mTORC1 activity in cells treated with the compounds. In
FIG. 20A, p-P70S6K (Thr389)/p-P85S6K (Thr412) level in MCF7 cells
is shown. In FIG. 20B, p-S6 (Ser240/244) level in MCF7 cells is
shown. In FIG. 20C, the p-4E-BP1 (Thr37/46) level in MCF cells is
shown. In FIG. 20D, PC-3 cells were treated with test compounds at
above indicated concentrations (FIG. 15) for 24 h. pS6 level was
enhanced in insulin (100 .mu.g/mL, 30 min) treated cells. Y Axis is
expressed as Mean.+-.SD if applicable.
[0155] FIGS. 21A-B show the induction of caspase activity in cells
treated with the compounds. FIG. 21A shows MV-4-11 cells, caspase
activity was monitored at 6, 24, 48 and 72 h, and maximal activity
was found at 24 h. Both EX2 and EX1 are more potent than vorinostat
in terms of induction of caspase activity. GDC0941 showed very weak
activity and potency. EC.sub.50=0.52, 0.50, 1.16, and 12.7 .mu.M
for EX1, EX2, vorinostat and GDC-0941, respectively. FIG. 21B shows
PC-3 cells monitored at 24, 48 and 72 h time points. Maximal
caspase activity was found at 48 h. Both EX1 and EX2 are more
potent than vorinostat in terms of induction of caspase activity.
GDC-0941 showed very weak activity in both tumor cells. Y Axis is
expressed as Mean.+-.SD if applicable.
[0156] FIGS. 22A-D show the compound induced death of MV-4-11
cells. FIG. 22A shows EX1, compound example 1. FIG. 22B shows EX2,
compound example 2. FIG. 22C shows GDC-0941. FIG. 22D shows cell
death at 48 h for EX1, EX2, GDC-0941, and vorinostat. The cell
death potency EC.sub.50 was estimated as 0.19, 0.069, 0.58 and 2.75
.mu.M for EX1, EX2, vorinostat and GDC-0941, respectively. Y Axis
is expressed as Mean.+-.SD if applicable.
[0157] FIGS. 23A-B show histone hyperacetylation in PC-3 tumors
treated with the compounds. FIG. 23A shows a Western blot analysis
of tumor tissues. The lanes and the concentrations used were as
follows:
TABLE-US-00001 Lane Sample 1/2 Vehicle at 3 h 3/4 Vorinostat at 3 h
5/6 EX1 at 1 h 7/8 EX1 at 2 h 9/A EX1 at 3 h B EX78 at 4 h
[0158] Significant histone hyperacetylation in PC-3 tumor was
observed in treated mice. FIG. 23B shows the digitized and
normalized Western blot analysis results. Y Axis is expressed as
Mean.+-.SEM if applicable.
[0159] FIGS. 24A-B show histone hyperacetylation in MV4-11 tumors
treated with the compounds. FIG. 24A shows Western blot analyses of
tumor tissues. The lanes and the concentrations used were as
follows:
TABLE-US-00002 Lane Sample 1 Vehicle at 3 h, PO 2 EX2 at 1 h, PO 3
EX2 at 2 h, PO 4/5/6 EX2 at 3 h, PO 7 EX2 at 2 h, IV 8 EX2 at 3 h,
IV 9 EX2 at 2 h, IP A EX78 at 1 h, IV B EX78 at 2 h, PO C EX78 at 3
h, PO D EX78 at 4 h, PO
Significant histone hyperacetylation in MV4-11 tumor was observed
in treated mice. FIG. 24B shows the digitized and normalized
Western blot analysis results. Y Axis is expressed as Mean.+-.SEM
if applicable.
[0160] FIGS. 25A-B show efficacy of the compounds in NCr nude mice
HepG2 xenograft model. FIG. 25A shows significant tumor growth
inhibition (TGI) was achieved from day 4. TGI=96%, p=0.0034 on day
18 after last dose of 3.sup.rd cycle. The treatment continued to
4.sup.th cycle while the mice of vehicle group were euthanized due
to the tumor burden on day 18. FIG. 25B shows that EX2 was well
tolerated in NCr nude mice, no significant toxicity was observed at
this dose level. Y Axis is expressed as Mean.+-.SEM if
applicable.
[0161] FIGS. 26A-D show efficacy of the compounds in CB17 scid mice
HepG2 xenograft model. FIG. 26A shows that significant tumor growth
inhibition (TGI) was achieved from day 4. After last dose of
4.sup.th cycles, on day 26, TGI=117%, 82%, 38% and 98% for EX2
(150, 75 and 37.5 mg/kg) and sorafenib tosylate, respectively. FIG.
26B shows that on day 26, tumor size of treated group was
significantly smaller than vehicle group, with p<0.01 for EX2
150 and 75 mg/kg groups and sorafenib group, but p>0.05 for 37.5
mg/kg group. FIG. 26C shows that EX2 was well tolerated at all dose
levels, no significant body weight (BW) loss. Vehicle group has BW
loss due to increasing tumor burden. Sorafenib tosylate (100 mg/kg)
was not well tolerated; its dose was reduced to 80 mg/kg in the
3.sup.rd and 4.sup.th cycles. FIG. 26D shows that tumor size was
normalized against the initial value (as 100%). After last dose
(day 25), the tumor started to re-grow after a long period of
growth delay, but EX2 at 150 mg/kg seemed more effective than
sorafenib. Y Axis is expressed as Mean.+-.SEM if applicable.
[0162] FIGS. 27A-B show the efficacy of the compounds in NCr nude
mice HuH-7 xenograft model. FIG. 27A shows good tumor growth
inhibition with TGI=102% on day 12 after treatment of mice (mean
tumor volume 103 mm.sup.3 on day 0) with EX2 (QD.times.5.times.2).
FIG. 27B shows that EX2 was also effective on well-established
tumors (mean tumor size 363 mm.sup.3 on day 0) after one cycle of
treatment with TGI=88% (p=0.0016) and TGI=67% (p=0.0082) on day 12.
The tumor size was normalized against the initial volume (day 0 as
100%) in FIG. 27B. Y Axis is expressed as Mean.+-.SEM if
applicable.
[0163] FIGS. 28A-B show the efficacy of the compounds in 4T1 mouse
metastatic breast cancer model. In FIG. 28A, the tumor growth curve
is shown. In FIG. 28B, the tumor size on day 17 is shown, p=0.0063.
Y Axis is expressed as Mean.+-.SEM if applicable.
[0164] FIGS. 29A-B show the efficacy of the compounds in NCI-H460
lung cancer xenograft model. FIG. 29A shows that EX2 demonstrated
significant tumor growth inhibition with TGI=46% (p=0.0292) on day
12 after two cycles of treatment. FIG. 29B shows that EX2 was also
well tolerated at this dose level. Y Axis is expressed as
Mean.+-.SEM if applicable.
[0165] FIGS. 30A-B show the efficacy of the compounds in MV4-11
xenograft model. FIG. 30A shows the tumor growth curve: tumor size
was normalized against the initial value (day 0 as 100%), EX2
demonstrated significant tumor inhibition with average TGI=51%
between day 12 and day 20 (p<0.05) for 150 mg/kg group, but 75
mg/kg was not significantly effective. FIG. 30B shows the tumor
size on day 20, TGI=51% (p<0.05) and 20% (p>0.05) for 150 and
75 mg/kg, respectively. Y Axis is expressed as Mean.+-.SEM if
applicable.
[0166] FIG. 31 shows a compound of Formula (I), a family of fused
pyrimidine-based hydroxamate compounds.
DETAILED DESCRIPTION OF EMBODIMENTS
[0167] Hybrid-drug or multi-target drugs can increase the
probability of treatment or efficacy of treatment by acting on two
or more proven pathways or validated targets. For example, cancer
cell survival relies on many key pathways, thus, the blocking or
inhibition of one pathway may only have a small probability of
killing or inhibiting the growth of target cells. To illustrate, if
the probability of success is assumed to be 0.4 or 40% for each
pathway, the probability of failure is 1-0.4=0.6. If two pathways
(or targets) are targeted, the probability of failure will become
0.6.times.0.6=0.36, whilst the probability of success increased
drastically (1-0.36=0.64). If three pathways are targeted, the
chance of success will be 0.784 and increase so on and so
forth.
[0168] Biological systems are not simple, as they can compensate
each other and also synergize their functions. However, the
principle of targeting multiple pathways has been validated and is
already in use, such as in combination chemotherapy for cancer
treatment. Examples include combination of drugs such as histone
deacetylase (HDAC) inhibitors vorinostat and a variety of known
drugs in clinical trials, cocktail drugs for HIV treatment, and
augmentin (a mixture of amoxicillin and clavulanic acid) for
antibacterial treatment. There are also successful multi-target
drugs in the market, such as multi-kinase inhibitors sunitinib and
sorafenib. However, instead of using of two or more drugs for
combination or discovering some multi-target drug by chance, novel
multi-target drug molecules can be designed to target a combination
of validated and/or novel drug targets which work additively or
synergistically by incorporating the key chemical structure motifs
needed for each target and global modifying target profile and drug
like properties. The design and development of this type of drug
may be more challenging, but the advantage is that the molecules
are new chemical entities rather than physical mixtures or chemical
conjugates of two or more drugs, thus they are patentable and more
importantly, they have new pharmacological properties.
[0169] Epigenetics can be considered to be chemical modifications
to DNA which controls gene usage. Amino-acid residues of histone
molecules--especially those located at their amino (N)-terminal
tails--are subject to various post-translational modifications,
including methylation, acetylation, phosphorylation,
ubiquitination, sumoylation, citrullination and ADP ribosylation.
Several types of covalent modifications (such as acetylation and
lysine methylation) are reversible, whereas acetylation at various
residues is believed to have a more structural role, making the
nucleosome structure `looser` and more accessible to transcription
factors. Histone deacetylase (HDAC) has been a validated anticancer
drug target (FIG. 1) and research on HDAC inhibitors is ongoing. In
addition, HDAC inhibitors have potential use in autoimmune and
inflammatory disorders such as rheumatoid arthritis and diabetes
mellitus due to their ability to inhibit the expression of
pro-inflammatory cytokines such as TNF.alpha.. HDAC inhibitors may
also have non-oncology indications such as in self-renewal and
differentiation of stem cells.
[0170] HDAC inhibitors (HDACi) have been approved to be used
additively or synergistically with many chemotherapeutic agents and
kinase inhibitors. For example, treatment with HDACi panobinostat
combined with sorafenib demonstrated the highest preclinical
efficacy in treatment of hepatocellular carcinoma cancer (HCC)
models, providing the rationale for clinical studies with this
novel combination. mTOR inhibitors significantly enhanced
HDACi-induced apoptosis in HCC cells. The inhibition of both
mTORC1/2 not only efficiently blocked mTORC1 signalling, but also
abrogated AKT-feedback activation caused by selective mTORC1
inhibition. In vivo studies indicated that the combination of mTOR
inhibitor AZD8055 and HDACi vorinostat almost completely inhibited
tumor-growth, without obvious adverse effects, which suggested that
a combining-regimen of mTOR inhibitor and HDACi may be an effective
therapeutic strategy for treatment of HCC. Furthermore, the dual
PI3K/mTOR inhibitor PKI-587 (PF-05212384) and sorafenib targeting
PI3K/AKT/mTOR and Ras/Raf/MAPK pathways has been shown to
synergistically inhibit HCC cell proliferation. Thus the
HDAC-kinase inhibitor, particularly an inhibitor that multiply
inhibits the HDAC/PI3K-Akt-mTOR pathway would serve the purpose of
the two or three individual agents for the treatment of HCC and
other applicable diseases, provided that both the HDAC motif and
kinase scaffold are selected appropriately.
[0171] Imatinib is the first kinase that enjoyed great success in
both the field of science and sales. Since then, the study of
kinase inhibitors has become a very attractive field, and many
kinase drug candidates are now in clinical trials. The
phosphatidylinositide 3-kinase-AKT-mammalian target of the
rapamycin (PI3K-Akt-mTOR) signalling pathway widely regulates
divergent physiological processes and is crucial to many aspects of
cell growth and survival, including cell cycle progression,
differentiation, transcription, translation and apoptosis.
Dysregulation, either through amplification or as a direct result
of mutations, has been closely linked to the development and
progression of a wide range of cancers, prompting intense interest
in the development of small molecule modulators of key proteins in
this cascade. PI3K, Akt and other kinases, such as
3-phosphoinositide dependent protein kinase-1 (PDK1), mTOR, have
been directly targeted with varying degrees of clinical success to
date (FIG. 2).
[0172] PI3K-mTOR pathway also plays an import role in cell
migration and angiogenesis. The unique function of p110.alpha. in
regulating endothelial cell motility supports the importance of
this protein over p110.beta. and p110.delta. in vascular remodeling
and angiogenesis. Class II PI3K isoform, PI3K-C2.alpha. has a
crucial role in vascular formation and barrier integrity and
represents a new therapeutic target for vascular disease.
Temsirolimus, an mTOR inhibitor approved for the treatment of renal
cell carcinoma (RCC), inhibits proliferation and migration in
retinal pigment epithelial and endothelial cells via mTOR
Inhibition and decreases VEGF and PDGF Expression. CCI-779 Inhibits
rhabdomyosarcoma xenograft growth by an antiangiogenic mechanism
linked to the targeting of mTOR/Hif-1.alpha./VEGF Signaling. A
HDAC/PI3K-Akt-mTOR pathway multi-target inhibitor would be
beneficial for the treatment of hypervascular tumors such as HCC,
RCC and thyroid carcinomas as well as retinal angiogenesis
diseases.
[0173] Angiogenesis inhibitors have been successfully used in the
treatment of cancers. HDAC inhibitors are used to target tumor
angiogenesis, as they can alter vascular endothelial growth factor
signalling. Class IIb HDAC6 can regulate endothelial cell migration
and angiogenesis by deacetylation of cortactin and regulate cell
migration in an EB1-dependent manner. HDAC6 is therefore a target
for inhibiting endothelial cell migration and angiogenesis.
[0174] Both liver and kidney fibrosis have high unmet medical
needs. HDAC inhibitors have been studied in experimental liver and
kidney fibrosis. Histone deacetylase 2 is upregulated in normal and
keloid scars. Class II HDAC Inhibition hampers hepatic stellate
sell activation by induction of microRNA-29 and microRNA-29b
prevents liver fibrosis by attenuating hepatic stellate cell
activation and inducing apoptosis through targeting PI3K/AKT
pathway. Furthermore, HS-173, a novel PI3K inhibitor, attenuates
the activation of hepatic stellate cells in liver fibrosis. All
these growing evidences support development of a HDAC/PI3K-Akt-mTOR
pathway multi-target inhibitor for treatment of pathological
fibrosis.
[0175] A compound of Formula (I);
##STR00003##
[0176] wherein X, Y and Z may be independently selected from N,
CHR.sup.3 or CR.sup.3, wherein at least one of X, Y or Z is N;
[0177] may be a single or double bond, as valency allows;
[0178] R.sup.1 and R.sup.2 may be independently selected from the
group consisting of a bond, halogen, optionally substituted alkyl,
optionally substituted amino, optionally substituted alkyloxy,
optionally substituted cycloalkyl, optionally substituted
heterocycloalkyl, optionally substituted aryl and optionally
substituted heteroaryl;
[0179] R.sup.3 and R.sup.4 may be independently selected from the
group consisting of a bond, hydrogen, halogen, optionally
substituted alkyl, optionally substituted amino, optionally
substituted alkyloxy, optionally substituted cycloalkyl, optionally
substituted heterocycloalkyl, optionally substituted aryl and
optionally substituted heteroaryl;
[0180] at least one of R.sup.1, R.sup.2, R.sup.3 or R.sup.4 may be
further independently substituted by an hydroxamate group
-L.sup.1-R.sup.5-L.sup.2-R.sup.6-L.sup.3-CON(R.sup.a)OR.sup.b,
wherein;
[0181] R.sup.a and R.sup.b may be independently selected from the
group consisting of a bond, hydrogen, optionally substituted alkyl,
optionally substituted acyl and optionally substituted amino acid
residue;
[0182] L.sup.1, L.sup.2 and L.sup.3 may be independently selected
from the group consisting of a bond, optionally substituted alkyl,
optionally substituted alkenyl and optionally substituted
alkynyl;
[0183] R.sup.5 and R.sup.6 may be independently selected from the
group consisting of a bond, O, S, NR.sup.c, S(O).sub.n, optionally
substituted amide, optionally substituted urea, optionally
substituted carbonylurea, optionally substituted thiourea,
optionally substituted sulfonamide, optionally substituted
aminosulfonamide, optionally substituted sulfonylurea, optionally
substituted oxime, optionally substituted cycloalkyl, optionally
substituted heterocycloalkyl, optionally substituted aryl and
optionally substituted heteroaryl; wherein; [0184] R.sup.c may be
independently selected from the group consisting of a bond,
hydrogen, optionally substituted alkyl, optionally substituted
alkenyl, optionally substituted alkynyl and optionally substituted
acyl; and [0185] n may be an integer from 0 to 2;
[0186] or a pharmaceutically acceptable form or prodrug thereof, is
provided.
[0187] X, Y and Z may be independently selected from N, CHR.sup.3
or CR.sup.3, wherein at least one of X, Y or Z is N. X may be N,
CHR.sup.3 or CR.sup.3. Y may be N, CHR.sup.3 or CR.sup.3. Z may be
N, CHR.sup.3 or CR.sup.3. X, Y and Z may all be N. X and Z may both
be N and Y may be CR.sup.3. X may be CR.sup.3, Y may be CR.sup.3
and Z may be N. X may be CH.sub.2, Y may be CR.sup.3 and Z may be
N. X may be N, Y may be CR.sup.3 and Z may be CR.sup.3. X may be N,
Y may be CR.sup.3 and Z may be CH.
[0188] X may be CH.sub.2, Y may be CHR.sup.3 and Z may be N. X and
Z may both be N and Y may be CHR.sup.3. X may be CHR.sup.3, Y may
be CHR.sup.3 and Z may be N. X may be CH.sub.2, Y may be CHR.sup.3
and Z may be N. X may be N, Y may be CHR.sup.3 and Z may be
CHR.sup.3. X may be N, Y may be CHR.sup.3 and Z may be
CH.sub.2.
[0189] may be a single or double bond, as valency allows. X and Y
may be connected by a double bond. X and Y may be connected by a
single bond.
[0190] The compound may have any one of the following Formulae
(Ib), (Ic) or (Id);
##STR00004##
[0191] R.sup.1 and R.sup.2 may be independently selected from the
group consisting of a bond, halogen, optionally substituted alkyl,
optionally substituted amino, optionally substituted alkyloxy,
optionally substituted cycloalkyl, optionally substituted
heterocycloalkyl, optionally substituted aryl and optionally
substituted heteroaryl. R.sup.1 and R.sup.2 may be independently a
bond, halogen, optionally substituted amino, optionally substituted
alkylamino, optionally substituted cycloamino, optionally
substituted heterocycloamino, optionally substituted alkyl,
optionally substituted cycloalkyl, optionally substituted aryl or
optionally substituted heteroaryl. R.sup.1 and R.sup.2 may
independently a bond, halogen, optionally substituted amino,
optionally substituted alkylamino, optionally substituted
cycloamino, optionally substituted heterocycloamino, optionally
substituted arylamino, optionally substituted heteroarylamino,
optionally substituted alkyl, optionally substituted cycloalkyl,
optionally substituted aryl or optionally substituted heteroaryl.
R.sup.1 may be an optionally substituted heterocycloamino,
optionally substituted heteroaryl or optionally substituted
aryl.
[0192] R.sup.1 may be an optionally substituted phenyl, optionally
substituted pyrimidinyl, optionally substituted pyridinyl,
optionally substituted pyrazinyl, optionally substituted
thiomorpholino or optionally substituted morpholino.
[0193] R.sup.2 may be a halogen, optionally substituted amino,
optionally substituted alkylamino, optionally substituted
cycloamino, optionally substituted heterocycloamino, optionally
substituted aryl or optionally substituted heteroaryl. R.sup.2 may
be a Cl, Br, F, NH.sub.2, dimethylamino, diethylamino, optionally
substituted pyrrolidinyl, optionally substituted piperidinyl,
optionally substituted morpholino, optionally substituted phenyl,
optionally substituted pyridinyl, optionally substituted
pyrimidinyl, optionally substituted pyrazinyl, or optionally
substituted benzimidazolyl.
[0194] R.sup.3 and R.sup.4 may be independently selected from the
group consisting of a bond, hydrogen, halogen, optionally
substituted alkyl, optionally substituted amino, optionally
substituted alkyloxy, optionally substituted cycloalkyl, optionally
substituted heterocycloalkyl, optionally substituted aryl and
optionally substituted heteroaryl. R.sup.3 and R.sup.4 may be
independently a bond, hydrogen, optionally substituted alkyl,
optionally substituted amino, optionally substituted alkylamino,
optionally substituted cycloamino, optionally substituted
heterocycloamino, optionally substituted arylamino, optionally
substituted heteroarylamino, optionally substituted alkyloxy,
optionally substituted aryloxy, optionally substituted
heteroaryloxyl or optionally substituted cycloalkyl. R.sup.3 and
R.sup.4 may be independently a bond, hydrogen, optionally
substituted alkyl, optionally substituted amino, optionally
substituted alkylamino, optionally substituted cycloamino,
arylamino, optionally substituted heteroarylamino, optionally
substituted alkyloxy, optionally substituted aryloxy, optionally
substituted heteroaryloxyl or optionally substituted
cycloalkyl.
[0195] R.sup.3 may be a bond, hydrogen, optionally substituted
alkyl, optionally substituted amino, optionally substituted
alkylamino or optionally substituted cycloamino. R.sup.3 may be a
bond, hydrogen, NH.sub.2, diethylamino, optionally substituted
pyrrolidinyl or optionally substituted piperidinyl.
[0196] R.sup.4 may be a bond or optionally substituted alkyl.
R.sup.4 may be a bond, ethyl, 1-propyl, 2-propyl, 2-butyl, 3-pentyl
or cyclopentyl.
[0197] At least one of R.sup.1, R.sup.2, R.sup.3 or R.sup.4 may be
further independently substituted by an hydroxamate group
-L.sup.1-R.sup.5-L.sup.2-R.sup.6-L.sup.3-CON(R.sup.a)OR.sup.b.
[0198] R.sup.a and R.sup.b may be independently selected from the
group consisting of a bond, hydrogen, optionally substituted alkyl,
optionally substituted acyl and optionally substituted amino acid
residue. R.sup.a and R.sup.b may be hydrogen. The amino acid
residue may improve the solubility and bioavailability of the
prodrug.
[0199] L.sup.1, L.sup.2 and L.sup.3 may be independently selected
from the group consisting of a bond, optionally substituted alkyl,
optionally substituted alkenyl and optionally substituted alkynyl.
L.sup.1, L.sup.2 and L.sup.3 independently may have a carbon chain
length of C.sub.1 to C.sub.10.
[0200] R.sup.5 and R.sup.6 may be independently selected from the
group consisting of a bond, O, S, NR.sup.c, S(O).sub.n, optionally
substituted amide, optionally substituted urea, optionally
substituted carbonylurea, optionally substituted thiourea,
optionally substituted sulfonamide, optionally substituted
aminosulfonamide, optionally substituted sulfonylurea, optionally
substituted oxime, optionally substituted cycloalkyl, optionally
substituted heterocycloalkyl, optionally substituted aryl and
optionally substituted heteroaryl. R.sup.5 and R.sup.6 may be
independently a bond, --O--, --S--, --NH--, --N(Me)--, --N(Ac)--,
--S(O)--, --S(O).sub.2--, --CONH--, --NHCO--, --NHCONH--,
--S(O).sub.2NH--, NHS(O).sub.2--, --NHS(O).sub.2NH--, optionally
substituted heterocycloalkyl or optionally substituted aryl.
R.sup.5 and R.sup.6 may be independently a bond, --O--, --NH--,
--N(Me)--, --NHCO--, 1,3-piperidinylene, 1,4-piperidinylene,
2,4-pyrimidinylene, 2,5-pyrimidinylene, 1,2-phenylene,
1,3-phenylene or 1,4-phenylene.
[0201] R.sup.c may be independently selected from the group
consisting of a bond, hydrogen, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl and
optionally substituted acyl.
[0202] n may be an integer from 0 to 2.
[0203] The optionally substituted alkyl may be an optionally
substituted C.sub.1-C.sub.12 alkyl, optionally substituted
C.sub.1-C.sub.2 alkyl, optionally substituted C.sub.1-C.sub.4
alkyl, optionally substituted C.sub.2-C.sub.5 alkyl, optionally
substituted C.sub.1-C.sub.6 alkyl, optionally substituted
C.sub.1-C.sub.8 alkyl, optionally substituted C.sub.1-C.sub.10
alkyl, optionally substituted C.sub.2-C.sub.4 alkyl, optionally
substituted C.sub.2-C.sub.6 alkyl, optionally substituted
C.sub.2-C.sub.8 alkyl, optionally substituted C.sub.2-C.sub.10
alkyl, optionally substituted C.sub.1-C.sub.12 alkyl, substituted
C.sub.4-C.sub.6 alkyl, optionally substituted C.sub.4-C.sub.8
alkyl, optionally substituted C.sub.4-C.sub.10 alkyl, optionally
substituted C.sub.4-C.sub.12 alkyl, optionally substituted
C.sub.6-C.sub.8 alkyl, optionally substituted C.sub.6-C.sub.10
alkyl, optionally substituted C.sub.6-C.sub.12 alkyl, optionally
substituted C.sub.8-C.sub.10 alkyl, optionally substituted
C.sub.8-C.sub.12 alkyl, or optionally substituted C.sub.10-C.sub.12
alkyl. The optionally substituted alkyl may be an optionally
substituted C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6,
C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11 or C.sub.12
alkyl.
[0204] The optionally substituted alkyloxy may be an optionally
substituted C.sub.1-C.sub.16 alkyloxy, optionally substituted
C.sub.1-C.sub.2 alkoxy, optionally substituted C.sub.1-C.sub.4
alkoxy, optionally substituted C.sub.1-C.sub.6 alkoxy, optionally
substituted C.sub.1-C.sub.8 alkoxy, optionally substituted
C.sub.1-C.sub.10 alkoxy, optionally substituted C.sub.1-C.sub.12
alkoxy, optionally substituted C.sub.1-C.sub.14 alkoxy, optionally
substituted C.sub.2-C.sub.4 alkoxy, optionally substituted
C.sub.2-C.sub.6 alkoxy, optionally substituted C.sub.2-C.sub.8
alkoxy, optionally substituted C.sub.2-C.sub.10 alkoxy, optionally
substituted C.sub.2-C.sub.12 alkoxy, optionally substituted
C.sub.2-C.sub.14 alkoxy, optionally substituted C.sub.2-C.sub.16
alkoxy, optionally substituted C.sub.4-C.sub.6 alkoxy, optionally
substituted C.sub.4-C.sub.8 alkoxy, optionally substituted
C.sub.4-C.sub.10 alkoxy, optionally substituted C.sub.4-C.sub.12
alkoxy, optionally substituted C.sub.4-C.sub.14 alkoxy, optionally
substituted C.sub.4-C.sub.16 alkoxy, optionally substituted
C.sub.6-C.sub.8 alkoxy, optionally substituted C.sub.6-C.sub.10
alkoxy, optionally substituted C.sub.6-C.sub.12 alkoxy, optionally
substituted C.sub.6-C.sub.14 alkoxy, optionally substituted
C.sub.6-C.sub.16 alkoxy, optionally substituted C.sub.8-C.sub.10
alkoxy, optionally substituted C.sub.8-C.sub.12 alkoxy, optionally
substituted C.sub.8-C.sub.14 alkoxy, optionally substituted
C.sub.8-C.sub.16 alkoxy, optionally substituted C.sub.10-C.sub.12
alkoxy, optionally substituted C.sub.10-C.sub.14 alkoxy, optionally
substituted C.sub.10-C.sub.16 alkoxy, optionally substituted
C.sub.12-C.sub.14 alkoxy, optionally substituted C.sub.12-C.sub.16
alkoxy or optionally substituted C.sub.14-C.sub.16 alkoxy. The
optionally substituted alkoxy may be an optionally substituted
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.7,
C.sub.8, C.sub.9, C.sub.10, C.sub.11, C.sub.12, C.sub.13, C.sub.14,
C.sub.15 or C.sub.16 alkoxy.
[0205] The optionally substituted cycloalkyl may be an optionally
substituted C.sub.3-C.sub.9 cycloalkyl, optionally substituted
C.sub.3-C.sub.6 cycloalkyl or optionally substituted
C.sub.3-C.sub.9 cycloalkyl. The optionally substituted cycloalkyl
may be an optionally substituted C.sub.3, C.sub.4, C.sub.5,
C.sub.6, C.sub.7, C.sub.8 or C.sub.9 cycloalkyl.
[0206] The optionally substituted heterocycloalkyl may be an
optionally substituted heterocycloalkyl having a ring atom number
of 3 to 8, optionally substituted heterocycloalkyl having a ring
atom number of 3 to 4, an optionally substituted heterocycloalkyl
having a ring atom number of 3 to 5, an optionally substituted
heterocycloalkyl having a ring atom number of 3 to 6, an optionally
substituted heterocycloalkyl having a ring atom number of 3 to 7,
an optionally substituted heterocycloalkyl having a ring atom
number of 4 to 5, an optionally substituted heterocycloalkyl having
a ring atom number of 4 to 6, an optionally substituted
heterocycloalkyl having a ring atom number of 4 to 7, an optionally
substituted heterocycloalkyl having a ring atom number of 4 to 8,
an optionally substituted heterocycloalkyl having a ring atom
number of 5 to 6, an optionally substituted heterocycloalkyl having
a ring atom number of 5 to 7, an optionally substituted
heterocycloalkyl having a ring atom number of 5 to 8, an optionally
substituted heterocycloalkyl having a ring atom number of 6 to 7,
an optionally substituted heterocycloalkyl having a ring atom
number of 6 to 8 or an optionally substituted heterocycloalkyl
having a ring atom number of 7 to 8. The optionally substituted
heterocycloalkyl may be an optionally substituted have a ring atom
number of 3, 4, 5, 6, 7 or 8. The optionally substituted
heterocycloalkyl may have 1 to 3 heteroatoms independently selected
from the group consisting of N, O and S. The optionally substituted
heterocycloalkyl may have 1 to 2 heteroatoms independently selected
from the group consisting of N, O and S. The optionally substituted
heterocycloalkyl may have 2 to 3 heteroatoms independently selected
from the group consisting of N, O and S.
[0207] The optionally substituted aryl may be an optionally
substituted C.sub.6-C.sub.18 aryl, optionally substituted
C.sub.6-C.sub.10 aryl, optionally substituted C.sub.6-C.sub.12
aryl, optionally substituted C.sub.6-C.sub.14 aryl, optionally
substituted C.sub.6-C.sub.16 aryl, substituted C.sub.8-C.sub.10
aryl, optionally substituted C.sub.8-C.sub.12 aryl, optionally
substituted C.sub.8-C.sub.14 aryl, optionally substituted
C.sub.8-C.sub.16 aryl, optionally substituted C.sub.8-C.sub.18
aryl, optionally substituted C.sub.10-C.sub.12 aryl, optionally
substituted C.sub.10-C.sub.14 aryl, optionally substituted
C.sub.10-C.sub.16 aryl, optionally substituted C.sub.10-C.sub.18
aryl, optionally substituted C.sub.12-C.sub.14 aryl, optionally
substituted C.sub.12-C.sub.16 aryl, optionally substituted
C.sub.12-C.sub.18 aryl, optionally substituted C.sub.14-C.sub.16
aryl, optionally substituted C.sub.14-C.sub.18 aryl or optionally
substituted C.sub.14-C.sub.18 aryl. The optionally substituted aryl
may be an optionally substituted C.sub.6, C.sub.7, C.sub.8,
C.sub.9, C.sub.10, C.sub.11, C.sub.12, C.sub.13, C.sub.14,
C.sub.15, C.sub.16, C.sub.17 or C.sub.18 aryl.
[0208] The optionally substituted heteroaryl may be heteroaryl
having a ring atom number of 3 to 8, optionally substituted
heteroaryl having a ring atom number of 3 to 4, an optionally
substituted heteroaryl having a ring atom number of 3 to 5, an
optionally substituted heteroaryl having a ring atom number of 3 to
6, an optionally substituted heteroaryl having a ring atom number
of 3 to 7, an optionally substituted heteroaryl having a ring atom
number of 4 to 5, an optionally substituted heteroaryl having a
ring atom number of 4 to 6, an optionally substituted heteroaryl
having a ring atom number of 4 to 7, an optionally substituted
heteroaryl having a ring atom number of 4 to 8, an optionally
substituted heteroaryl having a ring atom number of 5 to 6, an
optionally substituted heteroaryl having a ring atom number of 5 to
7, an optionally substituted heteroaryl having a ring atom number
of 5 to 8, an optionally substituted heteroaryl having a ring atom
number of 6 to 7, an optionally substituted heteroaryl having a
ring atom number of 6 to 8 or an optionally substituted heteroaryl
having a ring atom number of 7 to 8. The optionally substituted
heteroaryl may be an optionally substituted have a ring atom number
of 3, 4, 5, 6, 7 or 8. The optionally substituted heteroaryl may
have 1 to 3 heteroatoms independently selected from the group
consisting of N, O and S. The optionally substituted heteroaryl may
have 1 to 2 heteroatoms independently selected from the group
consisting of N, O and S. The optionally substituted heteroaryl may
have 2 to 3 heteroatoms independently selected from the group of
consisting of N, O and S.
[0209] The optionally substituted alkenyl may be an optionally
substituted C.sub.2-C.sub.12 alkenyl, optionally substituted
C.sub.2-C.sub.4 alkenyl, optionally substituted C.sub.2-C.sub.6
alkenyl, optionally substituted C.sub.2-C.sub.8 alkenyl, optionally
substituted C.sub.2-C.sub.10 alkenyl, optionally substituted
C.sub.1-C.sub.12 alkenyl, substituted C.sub.4-C.sub.6 alkenyl,
optionally substituted C.sub.4-C.sub.8 alkenyl, optionally
substituted C.sub.4-C.sub.10 alkenyl, optionally substituted
C.sub.4-C.sub.12 alkenyl, optionally substituted C.sub.6-C.sub.8
alkenyl, optionally substituted C.sub.6-C.sub.10 alkenyl,
optionally substituted C.sub.6-C.sub.2 alkenyl, optionally
substituted C.sub.8-C.sub.10 alkenyl, optionally substituted
C.sub.8-C.sub.12 alkenyl, or optionally substituted
C.sub.10-C.sub.12 alkenyl. The optionally substituted alkenyl may
be an optionally substituted C.sub.2, C.sub.3, C.sub.4, C.sub.5,
C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11 or C.sub.12
alkenyl.
[0210] The optionally substituted alkynyl is an optionally
substituted C.sub.2-C.sub.12 alkynyl, optionally substituted
C.sub.2-C.sub.4 alkynyl, optionally substituted C.sub.2-C.sub.6
alkynyl, optionally substituted C.sub.2-C.sub.8 alkynyl, optionally
substituted C.sub.2-C.sub.10 alkynyl, optionally substituted
C.sub.1-C.sub.12 alkynyl, substituted C.sub.4-C.sub.6 alkynyl,
optionally substituted C.sub.4-C.sub.8 alkynyl, optionally
substituted C.sub.4-C.sub.10 alkynyl, optionally substituted
C.sub.4-C.sub.12 alkynyl, optionally substituted C.sub.6-C.sub.8
alkynyl, optionally substituted C.sub.6-C.sub.10 alkynyl,
optionally substituted C.sub.6-C.sub.12 alkynyl, optionally
substituted C.sub.8-C.sub.10 alkynyl, optionally substituted
C.sub.8-C.sub.12 alkynyl, or optionally substituted
C.sub.10-C.sub.12 alkynyl. The optionally substituted alkynyl may
be an optionally substituted C.sub.2, C.sub.3, C.sub.4, C.sub.5,
C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11 or C.sub.12
alkynyl.
[0211] The hydroxamate group
-L.sup.1-R.sup.5-L.sup.2-R.sup.6-L.sup.3-CON(R.sup.a)OR.sup.b may
be selected from any one of the following structures;
##STR00005##
[0212] R.sup.7 may be selected from the group consisting of a bond,
optionally substituted alkyl, optionally substituted aryl,
optionally substituted heteroaryl, O, S, optionally substituted
amino, --CH.sub.2O--, --OCH.sub.2--, --CH.sub.2S(O).sub.n--,
--S(O).sub.nCH.sub.2--, --S(O).sub.n--, --CH.sub.2N(R.sup.c)--,
--N(R.sup.c)CH.sub.2-, --N(R.sup.c).sup.-, --CO--,
--C(.dbd.NOR.sup.a)--, --CON(R.sup.a)--, --N(R.sup.c)CO--,
--N(R.sup.c)CON(R.sup.c)CO--, --CON(R.sup.c)CONH.sup.-,
--N(R.sup.c)CON(R.sub.b)--, S(O).sub.2N(R).sup.-,
--S(O).sub.2N(R.sup.a)CON(R)--,
--N(R.sup.c)CON(R.sup.a)S(O).sub.2--,
--N(R.sup.c)S(O).sub.2N(R.sup.a)-- and --N(R.sup.c)S(O).sub.2--;
and
[0213] m may be an integer from 0 to 10.
[0214] R.sup.2 or R.sup.4 may contain the hydroxamate group
-L.sup.1-R.sup.5-L.sup.2-R.sup.6-L.sup.3-CON(R.sup.a)OR.sup.bR.sup.1
may not be a morpholine when R.sup.2 or R.sup.3 contains the
hydroxamate group.
[0215] R.sup.1 may be a substituted amino when R.sup.2 or R.sup.3
contains the hydroxamate group. The substituted amino may be
morpholine.
[0216] The compound may have the following Formula (Ib):
##STR00006##
[0217] wherein R.sup.1 may be an optionally substituted phenyl,
optionally substituted pyrimidinyl, optionally substituted
pyridinyl, optionally substituted pyrazinyl, optionally substituted
thiomorpholino or optionally substituted morpholino;
[0218] R.sup.2 may be a Cl, Br, F, NH.sub.2, dimethylamino,
diethylamino, optionally substituted pyrrolidinyl, optionally
substituted piperidinyl, optionally substituted morpholino,
optionally substituted phenyl, optionally substituted pyridinyl,
optionally substituted pyrimidinyl, optionally substituted
pyrazinyl, or optionally substituted benzimidazolyl;
[0219] R.sup.3 may be a bond, hydrogen, NH.sub.2, diethylamino,
optionally substituted pyrrolidinyl or optionally substituted
piperidinyl; and
[0220] R.sup.4 may be a bond, ethyl, 1-propyl, 2-propyl, 2-butyl,
3-pentyl or cyclopentyl.
[0221] Specific compounds of the disclosure include the
following:
##STR00007## ##STR00008## ##STR00009## ##STR00010## ##STR00011##
##STR00012## ##STR00013## ##STR00014## ##STR00015## ##STR00016##
##STR00017## ##STR00018##
[0222] or a pharmaceutically acceptable salt or prodrug
thereof.
[0223] Specific compounds when R.sup.1 is not a morpholine when
R.sup.2 or R.sup.3 contains the hydroxamate group may include the
following:
##STR00019## ##STR00020## ##STR00021## ##STR00022## ##STR00023##
##STR00024## ##STR00025## ##STR00026##
[0224] or a pharmaceutically acceptable salt or prodrug
thereof.
[0225] Specific compounds wherein R.sup.1 is a substituted amino
when R.sup.2 or R.sup.3 contains the hydroxamate group and the
substituted amino is morpholine may include the following:
##STR00027## ##STR00028##
[0226] or a pharmaceutically acceptable salt or prodrug
thereof.
[0227] The compounds as defined above may be an enzyme inhibitor.
The compounds as defined above may have the ability to inhibit the
activity of certain deacetylases and protein kinases. The
deacetylase may be histone deacetylase. The ability to inhibit
deacetylase activity may be a result of the compounds acting
directly and solely on the histone deacetylase and/or non-histone
deacetylase molecule to inhibit biological activity. The kinase may
be a lipid kinase or a protein kinase. The kinase may be a lipid
kinase and a protein kinase. The kinase may be
phosphatidylinositate-3-kinase (PI3K). The ability to inhibit
kinase activity may be a result of the compounds acting directly
and solely on the kinase molecule to inhibit biological activity.
However, it is understood that the compounds may also act at least
partially on co-factors of the kinase in question that are involved
in the phosphorylation process.
[0228] The compounds disclosed herein may act directly and solely
on the deacetylase molecule or a complex or fragment thereof to
inhibit biological activity. However, it is understood that the
compounds may also act at least partially on co-factors that are
involved in the deacetylation process. Known kinase co-factors
include ionic species (such as zinc).
[0229] The compounds disclosed herein may act directly and solely
on the kinase molecule or a complex or fragment thereof to inhibit
biological activity. However, it is understood that the compounds
may also act at least partially on co-factors that are involved in
the phosphorylation process. Known kinase co-factors include ionic
species (such as zinc and calcium), lipids (such as
phosphatidylserine), and diacylglycerols.
[0230] The compounds as defined above may have activity against
HDACs and/or PI3K kinases or a fragment or a complex or a
functional equivalent thereof. The compounds as defined above may
be a histone deacetylase inhibitor (HDAC) or a
phosphatidylinositate-3-kinase (PI3K) inhibitor. The compounds as
defined above may be a histone deacetylase (HDAC) inhibitor and a
phosphatidylinositate-3-kinase (PI3K) inhibitor. The compounds as
defined above may be an inhibitor of the PI3K-AKT-mTOR pathway. The
compounds as defined above may be a multi-target inhibitor.
[0231] The compounds as defined above may inhibit histone
deacetylase (HDAC) and phosphatidylinositate-3-kinase (PI3K)
simultaneously.
[0232] The compounds as defined above may have activity against
certain serine/threonine kinases such as mTOR or Akt or a fragment
or complex or functional equivalent thereof.
[0233] The inhibition of the lipid kinase and protein kinase may be
carried out in any of a number of well-known ways in the art. For
example if inhibition of the protein kinase in vitro is desired, an
appropriate amount of the compound may be added to a solution
containing the kinase. In circumstances where it is desired to
inhibit the activity of the kinase in a mammal, the inhibition of
the kinase may typically involve administering the compound to a
mammal containing the kinase.
[0234] A method of inhibiting HDAC and/or PI3K in a cell may
comprise administering to a cell a compound as defined above, or a
pharmaceutically acceptable form or prodrug thereof. The inhibition
of HDAC and/or PI3K may further comprise the inhibition of cell
proliferation. The inhibition of HDAC and/or PI3K may further
comprise reprogramming cells to induce pluripotent stem cells (iPS
cells).
[0235] The cell may be in vitro. The cell may be from a cell line.
The cell line may be an immortalized cell line, a genetically
modified cell line or a primary cell line. The cell line may be
selected from the group consisting of MV4-11, MOLT-4, PC-3, MCF7,
SUP-B15, HL-60, K-562, RPMI-8226, Daudi, Raji, Ramos, Pfeiffer,
A431, ACHN, A549, COLO 205, HCT116, HEL92.1.7, NCI-H522, A375,
NCI-H460, BxPC-3, PANC-1, SK-OV-3, U87MG, U138MG, HpeG2, SK-HEP1,
HuH-7, HCCLM3, PLC/PRF/5, HeLa, BT 474, MDA-MB-231, MDA-MB-436 and
MDA-MB-468.
[0236] The cell may be from tissue of a subject. The cell may be in
a subject.
[0237] These compounds as defined above may be used as modulators
or inhibitors for oncology indications as well as non-oncology
indications and applications such as autoimmune and inflammatory
disorders, self-renewal and differentiation of stem cells.
[0238] Accordingly the compounds as defined above may find a
multiple number of applications in which their ability to inhibit
lipid and protein kinases of the type mentioned above can be
utilised. For example the compounds as defined above may be used to
inhibit serine/threonine protein kinases. The compounds may also be
used in treating or preventing a condition in a mammal in which
inhibition of a protein kinase and/or co-factor thereof prevents,
inhibits or ameliorates a pathology or a symptomology of the
condition.
[0239] The compound as defined above, or a pharmaceutically form or
prodrug thereof, or a composition as defined above, may be for use
in therapy.
[0240] A method of treating a HDAC- or PI3K-related disorder may
comprise administering to a subject in need of treatment a compound
as defined above, or a pharmaceutically acceptable form or prodrug
thereof, or a composition as defined above. A method of treating a
HDAC- and PI3K-related disorder may comprise administering to a
subject in need of treatment a compound as defined above, or a
pharmaceutically acceptable form or prodrug thereof, or a
composition as defined above.
[0241] The method may further comprise the step of administering an
additional therapeutic agent in the subject. A method of modulating
the self-renewal or differentiation of stem-cells may comprise
administering to a subject in need of treatment a compound as
defined above, or a pharmaceutically acceptable form or prodrug
thereof, or a composition as defined above.
[0242] The compounds as defined above may also be used in the
preparation of a medicament for treating a condition in an animal
in which inhibition of a protein kinase can prevent, inhibit or
ameliorate the pathology or symptomology of the condition. The
compounds as defined above may also be used in the preparation of a
medicament for the treatment or prevention of a kinase-related
disorder.
[0243] A use of a compound as defined above, or a pharmaceutically
acceptable form or prodrug thereof, or a composition according as
defined above, may be in the manufacture of a medicament for
treatment of a HDAC- or PI3K-related disorder. A use of a compound
as defined above, or a pharmaceutically acceptable form or prodrug
thereof, or a composition as defined above, may be in the
manufacture of a medicament for treatment of a HDAC- and
PI3K-related disorder.
[0244] A use of the compound as defined above, or a
pharmaceutically acceptable form or prodrug thereof, or a
composition as defined above, may be in the manufacture of a
medicament for modulating the self-renewal or differentiation of
stem-cells.
[0245] The use may further comprise the medicament to be
administered with an additional therapeutic agent, wherein said
medicament may be administered in combination or alteration with
the additional therapeutic agent.
[0246] The conditions or disorders may be selected from the group
consisting of cancer, angiogenic disorder or pathological
angiogenesis, fibrosis, inflammatory conditions, asthma,
neurological disorders, neurodegenerative disorders, muscle
degenerative disorders, autoimmune disorders, disorders of the
blood or disorders of the bone marrow. The condition or disorder
may be lymphoma, cutaneous T-cell lymphoma, follicular lymphoma, or
Hodgkin lymphoma, cervical cancer, ovarian cancer, breast cancer,
lung cancer, prostate cancer, colorectal cancer, sarcoma,
hepatocellular carcinoma, leukemia or myeloma, retinal angiogenic
disease, liver fibrosis, kidney fibrosis, Alzheimer's disease or
Huntington's disease, spinal muscular atrophy, HIV/AIDS,
polycythemia vera or essential thrombocythemia or
myelofibrosis.
[0247] It is anticipated that the compounds as defined above will
be useful in treating various cancers including but not limited to
bone cancers, brain and CNS tumours, breast cancers, colorectal
cancers, endocrine cancers including adrenocortical carcinoma,
pancreatic cancer, pituitary cancer, thyroid cancer, parathyroid
cancer, thymus cancer, gastrointestinal cancers, liver cancer,
extra hepatic bile duct cancer, gastrointestinal carcinoid tumour,
gall bladder cancer, genitourinary cancers, gynaecological cancers,
head and neck cancers, leukemias, myelomas, hematological
disorders, lung cancers, lymphomas, eye cancers, skin cancers, soft
tissue sarcomas, adult soft tissue sarcoma, Kaposi's sarcoma,
urinary system cancers.
[0248] Exemplary cancers that may be treated by the compounds as
defined above include hematologic cancer and solid tumor such as
myeloproliferative disorders (idiopathic myelofibrosis,
polycythemia vera, essential thrombocythemia, chronic myeloid
leukemia), myeloid metaplasia, chronic myelomonocytic leukemia,
acute lymphocytic leukemia, acute erythroblastic leukemia,
Hodgkin's and Non Hodgkin's disease, B-cell lymphoma, diffuse large
B cell lymphoma, acute T-cell leukemia, myelodysplastic syndromes,
plasma cell disorder, hairy cell leukemia, kaposi's sarcoma,
lymphoma; gynaecologic cancer such as breast carcinoma, ovarian
cancer, cervical cancer, vaginal and vulva cancer, endometrial
hyperplasia; gastrointestinal tract cancer such as colorectal
carcinoma, polyps, liver cancer, gastric cancer, pancreatic cancer,
gall bladder cancer; urinary tract cancer such as prostate cancer,
kidney and renal cancer; urinary bladder cancer, urethral cancer,
penile cancer; skin cancer such as melanoma; brain tumour such as
glioblastoma, neuroblastoma, astrocytoma, ependynoma, brain-stem
gliomas, medulloblastoma, menigiomas, astrocytoma,
oligodendroglioma; head and neck cancer such as nasopharyngeal
carcinoma, laryngeal carcinoma; respiratory tract cancer such as
lung carcinoma (NSCLC and SCLC), mesothelioma; eye disease such as
retinoblastoma; musculo-skeleton diseases such as osteosarcoma,
musculoskeleletal neoplasm; Squamous cell carcinoma and fibroid
tumour.
[0249] Administration of compounds as defined above to humans may
be done by any of the accepted modes for enteral administration
such as oral or rectal, or by parenteral administration such as
subcutaneous, intramuscular, intravenous and intradermal routes.
Injection may be bolus or via constant or intermittent infusion.
The active compound as defined above may typically be included in a
pharmaceutically acceptable carrier or diluent and in an amount
sufficient to deliver to the patient at a therapeutically effective
dose. In various embodiments, the inhibitor compound may be
selectively toxic or more toxic to rapidly proliferating cells,
e.g. cancerous tumours, than to normal cells.
[0250] In using the compounds as defined above, they may be
administered in any form or mode which makes the compound
bioavailable. One skilled in the art of preparing formulations can
readily select the proper form and mode of administration depending
upon the particular characteristics of the compound selected, the
condition to be treated, the stage of the condition to be treated
and other relevant circumstances.
[0251] The compounds as defined above may be administered alone or
in the form of a pharmaceutical composition in combination with a
pharmaceutically acceptable carrier, diluent or excipient. The
compounds, while effective themselves, are typically formulated and
administered in the form of their pharmaceutically acceptable salts
as these forms are typically more stable, more easily crystallised
and have increased solubility.
[0252] The compounds as defined above are, however, typically used
in the form of pharmaceutical compositions which are formulated
depending on the desired mode of administration. A pharmaceutical
composition may comprise a compound as defined above, or a
pharmaceutically acceptable form or prodrug thereof, and a
pharmaceutically acceptable excipient. As such in some embodiments
the present disclosure provides a pharmaceutical composition
including a compound of Formula (I) and a pharmaceutically
acceptable carrier, diluent or excipient. The compositions may be
prepared in manners well known in the art.
[0253] In other embodiments there is provided a pharmaceutical pack
or kit comprising one or more containers filled with one or more of
the ingredients of the pharmaceutical compositions. In such a pack
or kit, a container having a unit dosage of the agent (s) may be
found. The kits may include a composition comprising an effective
agent either as concentrates (including lyophilized compositions),
which may be diluted further prior to use or they can be provided
at the concentration of use, where the vials may include one or
more dosages. Conveniently, in the kits, single dosages can be
provided in sterile vials so that the physician can employ the
vials directly, where the vials will have the desired amount and
concentration of agent(s). Associated with such container(s) may be
various written materials such as instructions for use, or a notice
in the form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
[0254] The compounds may be used or administered in combination
with one or more additional drug(s) for the treatment of the
disorder/diseases mentioned. The components may be administered in
the same formulation or in separate formulations. If administered
in separate formulations the compounds may be administered
sequentially or simultaneously with the other drug(s).
[0255] In addition to being able to be administered in combination
with one or more additional drugs, the compounds may be used in a
combination therapy. When this is done the compounds are typically
administered in combination with each other. Thus one or more of
the compounds may be administered either simultaneously (as a
combined preparation) or sequentially in order to achieve a desired
effect. This is especially desirable where the therapeutic profile
of each compound is different such that the combined effect of the
two drugs provides an improved therapeutic result.
[0256] Pharmaceutical compositions for parenteral injection may
comprise pharmaceutically acceptable sterile aqueous or nonaqueous
solutions, dispersions, suspensions or emulsions as well as sterile
powders for reconstitution into sterile injectable solutions or
dispersions just prior to use. Examples of suitable aqueous and
nonaqueous carriers, diluents, solvents or vehicles include water,
ethanol, polyols (such as glycerol, propylene glycol, polyethylene
glycol, and the like), and suitable mixtures thereof, vegetable
oils (such as olive oil), and injectable organic esters such as
ethyl oleate. Proper fluidity may be maintained, for example, by
the use of coating materials such as lecithin, by the maintenance
of the required particle size in the case of dispersions, and by
the use of surfactants.
[0257] These compositions may also contain adjuvants such as
preservative, wetting agents, emulsifying agents, and dispersing
agents. Prevention of the action of micro-organisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents such as
sugars, sodium chloride, and the like. Prolonged absorption of the
injectable pharmaceutical form may be brought about by the
inclusion of agents that delay absorption such as aluminium
monostearate and gelatin.
[0258] If desired, and for more effective distribution, the
compounds may be incorporated into slow release or targeted
delivery systems such as polymer matrices, liposomes, and
microspheres.
[0259] The injectable formulations may be sterilized, for example,
by filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions that can be dissolved or dispersed in sterile water or
other sterile injectable medium just prior to use.
[0260] Solid dosage forms for oral administration may include
capsules, tablets, pills, powders, and granules. In such solid
dosage forms, the active compound may be mixed with at least one
inert, pharmaceutically acceptable excipient or carrier such as
sodium citrate or dicalcium phosphate and/or a) fillers or
extenders such as starches, lactose, sucrose, glucose, mannitol,
and silicic acid, b) binders such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,
sucrose, and acacia, c) humectants such as glycerol, d)
disintegrating agents such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate, e) solution retarding agents such as paraffin, f)
absorption accelerators such as quaternary ammonium compounds, g)
wetting agents such as, for example, cetyl alcohol and glycerol
monostearate, h) absorbents such as kaolin and bentonite clay, and
i) lubricants such as talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof. In the case of capsules, tablets and pills, the dosage
form may also comprise buffering agents.
[0261] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like.
[0262] The solid dosage forms of tablets, dragees, capsules, pills,
and granules may be prepared with coatings and shells such as
enteric coatings and other coatings well known in the
pharmaceutical formulating art. They may optionally contain
opacifying agents and can also be of a composition that they
release the active ingredient(s) only, or preferentially, in a
certain part of the intestinal tract, optionally, in a delayed
manner. Examples of embedding compositions which may be used
include polymeric substances and waxes.
[0263] The active compounds may also be in microencapsulated form,
if appropriate, with one or more of the above-mentioned
excipients.
[0264] Liquid dosage forms for oral administration may include
pharmaceutically acceptable emulsions, solutions, suspensions,
syrups and elixirs. In addition to the active compounds, the liquid
dosage forms may contain inert diluents commonly used in the art
such as, for example, water or other solvents, solubilizing agents
and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethyl formamide, dimethyl
sulfoxide, oils (in particular, cottonseed, groundnut, corn, germ,
olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl
alcohol, Solutol.RTM. HS 15, Cremophor EL, polyethylene glycols and
fatty acid esters of sorbitan, and mixtures thereof.
[0265] Besides inert diluents, the oral compositions may also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, and perfuming agents.
[0266] Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminium metahydroxide, bentonite,
agar-agar, and tragacanth, and mixtures thereof.
[0267] Compositions for rectal or vaginal administration are
preferably suppositories which may be prepared by mixing the
compounds with suitable non-irritating excipients or carriers such
as cocoa butter, polyethylene glycol or a suppository wax which are
solid at room temperature but liquid at body temperature and
therefore melt in the rectum or vaginal cavity and release the
active compound.
[0268] Dosage forms for topical administration of a compound
include powders, patches, sprays, ointments and inhalants. The
active compound may be mixed under sterile conditions with a
pharmaceutically acceptable carrier and any needed preservatives,
buffers, or propellants which may be required.
[0269] The amount of compound administered will preferably treat
and reduce or alleviate the condition. A therapeutically effective
amount may be readily determined by an attending diagnostician by
the use of conventional techniques and by observing results
obtained under analogous circumstances. In determining the
therapeutically effective amount, a number of factors are to be
considered including but not limited to, the species of animal, its
size, age and general health, the specific condition involved, the
severity of the condition, the response of the patient to
treatment, the particular compound administered, the mode of
administration, the bioavailability of the preparation
administered, the dose regime selected, the use of other
medications and other relevant circumstances.
[0270] A preferred dosage may be a range from about 0.01 to 400 mg
per kilogram of body weight per day. A more preferred dosage may be
in the range from 0.1 to 200 mg per kilogram of body weight per
day, more preferably from 0.2 to 100 mg per kilogram of body weight
per day, even more preferably 0.2 to 50 mg per kilogram of body
weight per day. A suitable dose may be administered in multiple
sub-doses per day.
[0271] The process for synthesizing the compound of formula (I) may
comprise the steps of; (a) providing a halogen-disubstituted
purine-based or halogen di-substituted fused pyrimidine-based
compound; (b) alkylating the amine (--NH-- group) in the compound
of step (a); (c) selectively or sequentially displacing the halide
atoms of the intermediary compound of step (b) with an optionally
substituted boronic ester or an optionally substituted amine to
form a substituted aromatic or a substituted amine, respectively;
(d) selectively coupling the intermediary compound of step (c) with
a protected hydroxamic acid group having the structure
-L.sup.1-R.sup.5-L.sup.2-R.sup.6-L.sup.3-CON(R.sup.a)OR.sup.b or an
ester (hydroxamic acid precursor); and (e) converting the protected
hydroxamate or the ester of the intermediary compound of step (d)
to a hydroxamic acid under reaction conditions to form the compound
of formula (I).
[0272] A process for synthesizing the compound of formula (I) may
comprise the steps of; (a) providing a halogen-disubstituted
purine-based or halogen di-substituted fused pyrimidine-based
compound; (b) selectively displacing one of the halide atoms of
said compound with an optionally substituted boronic ester or an
optionally substituted amine to form a substituted aromatic or a
substituted amine, respectively; (c) alkylating the amine (--NH--
group) in the intermediary compound of step (b); (d) selectively
displacing the remaining halide atom of the intermediary compound
of step (c) with an optionally substituted boronic ester or an
optionally substituted amine to form a substituted aromatic or a
substituted amine, respectively; (e) selectively coupling the
intermediary compound of step (d) with a protected hydroxamic acid
group having the structure
-L.sup.1-R.sup.5-L.sup.2-R.sup.6-L.sup.3-CON(R.sup.a)OR.sup.b or an
ester (hydroxamic acid precursor); and (f) converting the protected
hydroxamate or the ester of the intermediary compound of step (e)
to a hydroxamic acid under reaction conditions to form the compound
of formula (I).
[0273] A process for synthesizing the compound of formula (I);
##STR00029##
[0274] may comprise the steps of; (a) providing a
halogen-disubstituted purine-based or halogen di-substituted fused
pyrimidine-based compound; (b) alkylating the amine in the compound
of step (a); (c) selectively or sequentially displacing the halide
atoms of the intermediary compound of step (b) with an optionally
substituted boronic ester or an optionally substituted amine to
form a substituted aromatic or a substituted amine, respectively;
(d) alkylating, in the intermediary compound of step (c), the
carbon atom that corresponds to the Y-position of formula (I); (e)
selectively coupling the intermediary compound of step (d) with a
protected hydroxamic acid group having the structure
-L.sup.1-R.sup.5-L.sup.2-R.sup.6-L.sup.3-CON(R.sup.a)OR.sup.b or an
ester (hydroxamic ester precursor); and (f) converting the
protected hydroxamate or the ester of the intermediary compound of
step (e) to a hydroxamic acid under reaction conditions to form the
compound of formula (I).
[0275] A process for synthesizing the compound of formula (I) may
comprise the steps of; (a)
[0276] providing a halogen-disubstituted purine-based or halogen
di-substituted fused pyrimidine-based compound; (b) selectively
displacing one of the halide atoms of said compound with an
optionally substituted boronic ester or an optionally substituted
amine to form a substituted aromatic or a substituted amine,
respectively; (c) alkylating the amine (--NH-- group) in the
intermediary compound of step (b); (d) alkylating, in the
intermediary compound of step (c), the carbon atom that corresponds
to the Y-position of formula (I); (e) selectively displacing the
remaining halide atom of the intermediary compound of step (d) with
an optionally substituted boronic ester or an optionally
substituted amine to form a substituted aromatic or a substituted
amine, respectively; (f) selectively coupling the compound of step
(e) with a protected hydroxamic acid group having the structure
-L.sup.1-R.sup.5-L.sup.2-R.sup.6-L.sup.3-CON(R.sup.a)OR.sup.b or an
ester (hydroxamic acid precursor); and (g) converting the protected
hydroxamate or the ester of the intermediary compound of step (f)
to a hydroxamic acid under reaction conditions to form the compound
of formula (I).
EXAMPLES
[0277] Non-limiting examples of the disclosure and a comparative
example will be further described in greater detail by reference to
specific Examples, which should not be construed as in any way
limiting the scope of the invention.
LIST OF ABBREVIATIONS USED
TABLE-US-00003 [0278] Names/terms Abbreviations Dichloroethane
(1,2-) DCE Dichloromethane DCM Dimethylformamide (N,N-) DMF
Dimethyl sulfoxide DMSO equivalent equiv High-performance liquid
HPLC chromatography or high- pressure liquid chromatography
high-resolution mass HRMS spectrometry N-Bromosuccinimide NBS
N-Methyl-2-pyrrolidone NMP Nuclear Magnetic Resonance NMR
Trifluoroacetic acid TFA Tetrahydrofuran THF
Example 1: Materials and Methods
[0279] In the examples described below, unless otherwise indicated,
all temperatures in the following description are in degrees
Celsius and all parts and percentages are by weight, unless
indicated otherwise. Reagents useful for synthesizing compounds may
be purchased from commercial suppliers, such as Sigma-Aldrich Pte
Ltd (Singapore 117528, Singapore), Boron Molecular Inc. (Raleigh,
N.C. 27616, USA), or Combi-Blocks, Inc. (San Diego, Calif. 92126,
USA), and used without further purification, unless otherwise
indicated, or obtained or prepared according to techniques known in
the art.
[0280] The reactions set forth below were performed under a
positive pressure of nitrogen, argon or with a drying tube, at
ambient temperature (unless otherwise stated), in anhydrous
solvents, and the reaction flasks are fitted with rubber septa for
the introduction of substrates and reagents via syringe. Glassware
was oven-dried and/or heat-dried. Analytical thin-layer
chromatography (TLC) was performed on glass-backed silica gel 60 F
254 plates (E Merck (0.25 mm)) and eluted with the appropriate
solvent ratios (v/v) and visualized by UV absorption. The reactions
were assayed by TLC and/or LC-MS and terminated as judged by the
consumption of starting material or the formation of desire
product.
[0281] Work-ups were typically done by doubling the reaction volume
with the reaction solvent or extraction solvent and then washing
with the indicated aqueous solutions using 25% by volume of the
extraction volume (unless otherwise indicated). Product solutions
were dried over anhydrous sodium sulphate (Na.sub.2SO.sub.4) or
magnesium sulphate (MgSO.sub.4) prior to filtration, and
evaporation of the solvents was under reduced pressure on a rotary
evaporator and noted as solvents removed in vacuo. Flash column
chromatography was conducted using silica gel 60 (Merck KGaA,
0.040-0.063 mm, 230-400 mesh ASTM).
[0282] Reverse-phase preparative high performance liquid
chromatography (RPHPLC) was conducted on a Gilson HPLC system
(331/332 pumps, GX-271 liquid handler, 172 diode array doctor
(DAD), Trilution LC software) using a Phenomenex column (Luna, 5
.mu.m, C18 100 A, 150 mm.times.21.2 mm) with adjustable solvent
gradients, usually 5-95% of acetonitrile in water+0.05% TFA in 15
or 20 min of gradient at flow rate of 20 mL/min, and was used for
routine purification. The preliminary purity and identity of all
compounds were assessed after purification by LC-MS analyses on a
Waters Micromass ZQ mass spectrometer in electrospray ionization
(ESI) positive mode after separation on a Waters 2795 separations
module. The HPLC separations were performed on a Phenomenex column
(Luna, 5 .mu.m, C18 100 A, 50 mm.times.2.00 mm) with a flow rate of
0.8 mL/min and a 4 min gradient of X-95% (X=5, 30 or 50) of
acetonitrile in water+0.05% TFA, using a Waters 2996 photodiode
array detector. Purity and identity were assessed on the integrated
UV chromatograms (220-400 nm) and the mass spectra. The final
purity was determined using a Shimadzu LC-20AD UFLC system on a
Phenomenex column (Luna, 5 .mu.m, C18 100 A, 50 mm.times.2.00 mm)
with a flow rate of 0.8 mL/min and a gradient of 5-95% of
acetonitrile in water+0.05% TFA over 6 min. All final products had
greater than 90% purity (by HPLC at wavelengths of 220 nm and 254
nm).
[0283] All the 1D and 2D NMR experiments for .sup.1H (400.13 MHz),
.sup.13C (100.61 MHz), .sup.15N (40.55 MHz), and .sup.19F (376.47
MHz) nuclei were performed on a Bruker AVANCE-400 digital NMR
spectrometer. NMR spectra are reported in ppm with reference to an
internal tetramethylsilane standard (0.00 ppm for .sup.1H and
.sup.13C) or solvent peak(s) of CDCl.sub.3 (7.26 and 77.1 ppm) or
CD.sub.3OD (3.31 and 49.0 ppm), or DMSO-d.sub.6 (2.50 and 39.5
ppm). Other NMR solvents were used as needed. When peak
multiplicities are reported, the following abbreviations are used:
s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet,
br=broadened, dd=doublet of doublets, dt=doublet of triplets,
bs=broadened singlet. Coupling constants, when given, are reported
in hertz.
[0284] Elemental analyses of CHN were performed on a Perkin-Elmer
2400 CHN/CHNS Elemental Analyzers. HRMS results were obtained from
a Bruker micrOTOF-Q II (ESI, positive mode) with direct injection
of purified compounds.
[0285] The agents of the various embodiments may be prepared using
the reaction routes and synthesis schemes as described below,
employing the techniques available in the art using starting
materials that are readily available. The preparation of particular
compounds of the embodiments is described in detail in the
following examples, but the artisan will recognize that the
chemical reactions described may be readily adapted to prepare a
number of other agents of the various embodiments. For example, the
synthesis of non-exemplified compounds may be successfully
performed by modifications apparent to those skilled in the art,
e.g. by appropriately protecting interfering groups, by changing to
other suitable reagents known in the art, or by making routine
modifications of reaction conditions. A list of suitable protecting
groups in organic synthesis can be found in both T. W. Greene and
P. G. Wuts' Protective Groups in Organic Synthesis, 3.sup.rd
Edition, John Wiley & Sons, New York, 1991 and P. J.
Kocienski's Protecting Groups, 3rd ed., Georg Thieme Verlag, New
York, 2005. Alternatively, other reactions disclosed herein or
known in the art will be recognized as having applicability for
preparing other compounds of the various embodiments.
Example 2: General Reaction Schemes
[0286] In practice, design and synthesis of a working multi-target
molecule by hybridising, merging or by de novo design is not a
simple task to achieve. The first step was to achieve dual
inhibition of both HDAC and kinases. HDAC inhibitor moieties were
introduced to a variety of positions to explore Structure Activity
Relationship (SAR) and the same was done for PI3K inhibition by
exploring a variety combination of groups for potency and isoform
selectivity. Substituent groups with a variety of properties,
including aromatic and non-aromatic, cyclic and acyclic, polar and
lipophilic, acidic, basic and neutral groups were used to cover the
SAR. As there is no known best HDAC/PI3K combination profile
available, the molecules were designed to have a broad range of
potency to achieve the best outcomes in the in vitro and in vivo
evaluations.
Scheme 1
[0287] A wide range of substituted purines and
pyrrolo[2,3-d]pyrimidines can be prepared in a straightforward
four- or five-step procedure starting from 2,6-dichloropurine or
2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine which are commercially
available from a number of sources. As depicted in FIG. 3, a
typical procedure uses an alkyl halide in the presence of a
suitable base such as potassium carbonate to alkylate the NH group
of compound 1. Alternatively, under Mitsunobu reaction conditions,
an alcohol may be reacted with 1 in the presence of a phosphine and
an activating agent, such as diethylazodicarboxylate, to afford a
similar alkylation product 2. The two chlorine atoms can be
displaced selectively or sequentially under optimal reaction
conditions. Under Suzuki reaction conditions, a boronic ester (4)
is used to replace the more active chlorine atom and form
mono-chloro compound 5. The 2.sup.nd chlorine atom can be displaced
by an amine (6), e.g., morpholine, at elevated temperature, in a
suitable solvent such as 1,4-dioxane, DMF, NMP, or THF, to give the
desired compound 7. Functional group P.sup.2 or R.sup.2b or P.sup.3
of 7 may contain an ester, a precursor of hydroxamic acid, or
protected hydroxamate, which can be easily converted to a
hydroxamic acid 8 or 9 or 12. If P.sup.3 group of 7 contains a
hydroxyl group (phenol or alcohol) or aldehyde, compound 7 can be
further varied by either alkylation of the hydroxyl group by a
halide (e.g., P.sup.5--P.sup.1, P.sup.1.dbd.Br) or reductive
amination of the aldehyde with an amine (e.g., P.sup.5--P.sup.1,
P.sup.1.dbd.--NH.sub.2 or --NH--) to afford 11 which is then
converted to hydroxamic acid 12 subsequently.
[0288] In FIG. 3 the reagents and conditions used are as follows:
(a) NaI/K.sub.2CO.sub.3 for P.sup.1.dbd.Br; (b) Mitsunobu reaction
for P.sup.1.dbd.OH, Ph.sub.3P/DEAD; (c) Suzuki coupling,
Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2, K.sub.2CO.sub.3/dioxane,
microwave irradiation or heat; (d) displacement; (e) NH.sub.2OH.HCl
(10 equiv)/NaOMe (20 equiv)/MeOH, 0.degree. C. to room temp; (f)
5-50% TFA/dichloromethane; (g) reductive amination, P.sup.5-P.sup.1
(10), P.sup.1.dbd.--NH-- or --NH.sub.2 and P.sup.3 containing
--CHO, NaBH(OAc).sub.3, DCE, room temp.
Scheme 2
[0289] FIG. 4 depicts an alternative displacement sequence of the
two chlorine atoms in compound 1 or 3. The more active chlorine
atom is displaced first by an amine 6 to give 13 via 1 or 14 via 3.
The NH group of 13 is alkylated later to form 14. In case of
P.sup.2 group of 14 is a nitrile, the nitrile group is reduced to
an amine, the latter then converted to a further substituted amine
15 either by alkylation with a halide or reductive amination with
an aldehyde. Amine 15 is further varied by Suzuki coupling reaction
to afford 16 which is subsequently converted to hydroxamic acid 17.
There are two options for compound 18 which is also derived from
mono-chloro compound 14: if the P.sup.2 or P.sup.3 group contains
an ester or protected hydroxamate, then it will be converted to
hydroxamic acid 19 or 21; if the P.sup.3 group contains either a
hydroxyl group (phenol or alcohol) or aldehyde, it will be
processed similar to compound 7 in Scheme 1, i.e., either
alkylation of the hydroxyl or reductive amination of the aldehyde
to form 20 which is subsequently converted to hydroxamic acid
21.
[0290] The reagents and conditions used in FIG. 4 are as follows:
(a) displacement, amine (6) neat or in dioxane, heat; (b)
K.sub.2CO.sub.3/DMF for P.sup.1=halide; c) Mitsunobu reaction for
P.sup.1.dbd.OH, Ph.sub.3P/DEAD; (d) P.sup.2 contains a terminal
nitrile group: reduction, NaBH.sub.4/NiCl.sub.2, THF-MeOH (1:2),
room temp.; (e) alkylation with P.sup.2CH.sub.2P.sup.1, P.sup.1 is
halide or leaving group; (f) reductive amination with P.sup.7CHO,
NaBH(OAc).sub.3, DCE, room temp.; (g) Suzuki coupling,
Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2, K.sub.2CO.sub.3/dioxane,
microwave or heat; (h) NH.sub.2OH.HCl (10 equiv)/NaOMe (20
equiv)/MeOH, -20.degree. C. to room temp; (i) 5-50%
TFA/dichloromethane
Scheme 3
[0291] FIG. 5 depicts how to further vary R.sup.3 group of compound
7 (scheme 1) and 18 (scheme 2) when R.sup.3 is a hydrogen and X is
a nitrogen. Both purines 7 and 18 are brominated with NBS to afford
bromides 22 and 26 respectively. The bromine atom at position 8 is
then displaced by an amine. An ester or protected hydroxamic acid
may exist either in P.sup.2 or R.sup.3b group of 23 and 27, thus
four types of hydroxamic acids 24, 25, 27 and 28 may form after
subsequent transformation.
[0292] In FIG. 5, the reagents and conditions used are as follows:
(a) NBS, CHCl.sub.3; (b) NMP or DMSO, 130.degree. C., 12 h; (c)
NH.sub.2OH.HCl (10 equiv)/NaOMe (20 equiv)/MeOH, -20.degree. C. to
room temp.
Scheme 4
[0293] FIG. 6 depicts an alternative method to vary R.sup.3 group
of compound 14 (scheme 2) when R.sup.3 is a hydrogen and X is a
nitrogen. Firstly, the position 8 of purine 14 is introduced an
aldehyde 29, then the latter is transformed to an amine 30. If the
R.sup.f group of 30 contains an ester or protected hydroxamate, it
will be converted to hydroxamic acid 32 via Suzuki product 31. When
30 is a secondary amine (R.sup.f.dbd.H), it can be further varied
by either alkylation with a halide or reductive amination with an
aldehyde to afford amine 33. Monochloroamine 33 is finally
converted to hydroxamic acid 35 via Suzuki reaction product 34.
[0294] In FIG. 6, the reagents and conditions used are as follows:
(a) DMF, n-BuLi; b) reductive amination with amine R.sup.dNH.sub.2,
NaBH.sub.4, DCE-MeOH; (c) Suzuki coupling,
Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2, K.sub.2CO.sub.3/dioxane,
microwave or heat; (d) NH.sub.2OH.HCl (10 equiv)/NaOMe (20
equiv)/MeOH, 0.degree. C. to room temp; (e) 5-50% TFA/DCM; (f)
alkylation with P.sup.12CH.sub.2P.sup.1, P.sup.1 is halide or
leaving group, Et.sub.3N; (g) reductive amination with P.sup.12CHO,
NaBH(OAc).sub.3, DCE, room temp.;
Scheme 5
[0295] FIG. 7 depicts the general synthetic routes for further
derivatization of readily or commercially available starting
materials (e.g., 4) and procedures for preparation of protected or
substituted hydroxamic acid (38). Boronic ester (4) is the key
material for Suzuki coupling reactions used by both Schemes 1 and
2, it can be modified prior to subjecting to Suzuki reaction. For
example, the amino group of boronic ester (4a) is coupled with an
acid to form an amide (4b) which has an ester group and is a
precursor for hydroxamic acid. Hydroxamic acid can be easily
generated from corresponding methyl or ethyl ester by treating it
with excessive hydroxylamine [i.e., NH.sub.2OH.HCl (10 equiv),
NaOMe (20 equiv) in MeOH]. Other methods of preparation of
protected or substituted hydroxylamine are described in Scheme 5.
An acid (37) can be converted to a hydroxamic acid (36) by
following methods. (i) the acid is converted to acid chloride under
milder conditions by treating it with ClCOCOCl, or SOCl.sub.2, or
other reagents under neutral conditions (such as Ph.sub.3P with
CBr.sub.4, or 2,4,6-Trichloro-[1,3,5]triazine); or (ii) the acid is
converted to an active ester by reacting it with isobutyl
chloroformate; (iii) the acid chloride or active ester is reacted
with hydroxylamine or the protected hydroxylamine R.sup.gNHOR.sup.h
[e.g., O-benzylhydroxylamine,
O-(2,4-dimethoxy-benzyl)-hydroxylamine,
O,N-bis-(2,4-dimethoxy-benzyl)-hydroxylamine,
O-(tetrahydro-pyran-2-yl)-hydroxylamine,
O-(tert-butyl-dimethyl-silyl)-hydroxylamine] to give the hydroxamic
acid or the protected hydroxamic acid; iv) coupling the acid with
hydroxylamine or protected hydroxylamine R.sup.gNHOR.sup.h with a
coupling reagent. The protecting group can be removed by methods
known in the literature such as hydrogenolysis to remove the
(substituted) benzyl group or acidic cleavage (e.g., TFA in DCM
with or without cation scavenger) to cleave the acid labile
protecting groups.
[0296] In FIG. 7, the reagents and conditions used are as follows:
(a) EDCI-HOBT/DCM; b) i) acid chloride formation ii) hydroxylamine
R.sup.iNHOR.sup.j; c) EDCI-HOBt, R.sup.iNHOR.sup.j.
Example 3: Synthesis of
N-hydroxy-4-(3-(9-isopropyl-2-morpholino-9H-purin-6-yl)phenoxy)butanamide
(FIG. 3, Compound 12a)
[0297] The reaction scheme for the synthesis of
N-hydroxy-4-(3-(9-isopropyl-2-morpholino-9H-purin-6-yl)phenoxy)butanamide
is shown in FIG. 8.
Step 1: Synthesis of 3-(2-chloro-9-isopropyl-9H-purin-6-yl)phenol
(5a)
[0298] To a pre-stirred of solution of
2,6-dichloro-9-isopropyl-9H-purine 3a (230 mg, 1.0 mmol),
(3-hydroxyphenyl)boronic acid 4c (152 mg, 1.1 mmol) in dioxane (10
mL), were added a solution of K.sub.2CO.sub.3 (345 mg, 2.5 mmol) in
deionized water (1.0 mL). The mixture was degassed for 30 min then
added Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2 (41 mg), and the resulting
mixture was heated at 70.degree. C. for 5 hours. LC-MS showed the
reaction completed. After a simple workup, the product 5a (215 mg,
75%) was obtained by flash chromatography (silica, 20% to 50% ethyl
acetate in hexanes).
Step 2: Synthesis of
3-(9-isopropyl-2-morpholino-9H-purin-6-yl)phenol (7a)
[0299] 3-(2-chloro-9-isopropyl-9H-purin-6-yl)phenol 5a (300 mg,
1.04 mmol) was dissolved in morpholine (5 mL). The resulting
mixture was heated at 80.degree. C. for 10 hours. LC-MS showed the
reaction completed. After a simple workup, the crude was purified
by flash chromatography (silica, ethyl acetate/hexanes=1:2) to
afford 7a (290 mg, 82%).
Step 3: Synthesis of ethyl
4-(3-(9-isopropyl-2-morpholino-9H-purin-6 yl)phenoxy)butanoate
(11a)
[0300] To a pre-stirred of solution of 7a (80 mg, 0.24 mmol), ethyl
6-bromobutyrate (69 mg, 0.35 mmol) in DMF (2 mL), was added
anhydrous potassium carbonate (98 mg, 0.79 mmol). The resulting
mixture was heated at 100.degree. C. overnight (16 h). After
workup, the crude was purified by flash chromatography (silica, 17%
to 25% of ethyl acetate in hexanes) to afford 11a (86 mg, 80%).
LC-MS m/z 454.2 ([M+H].sup.+). .sup.1HNMR (CDCl.sub.3) .delta. 8.32
(d, J=7.6 Hz, 1H), 8.20 (s, 1H), 7.96 (s, 1H), 7.43 (t, J=8.0 Hz,
1H), 7.02 (dd, J=8.0, 2.4 Hz, 1H), 4.80 (septet, J=6.8 Hz, 1H),
4.14 (m, 4H), 3.93 (t, J=4.8 Hz, 4H), 3.84 (t, J=4.8 Hz, 4H), 2.56
(t, J=7.4 Hz, 2H), 2.15 (quintet, J=6.8 Hz, 2H), 1.61 (d, J=6.8 Hz,
6H), 1.25 (t, J=7.2 Hz, 3H). .sup.13C NMR (CDCl.sub.3) .delta.
173.3, 159.0, 158.7, 154.4, 153.9, 139.2, 137.6, 129.5, 124.3,
122.3, 117.0, 115.0, 67.0, 66.7, 60.4, 46.8, 45.1, 30.8, 24.6,
23.4, 14.3.
Step 4: Synthesis of
N-hydroxy-4-(3-(9-isopropyl-2-morpholino-9H-purin-6-yl)phenoxy)butanamide
(12a)
[0301] To a pre-stirred of solution of 11a (55 mg, 0.12 mmol),
hydroxylamine hydrochloride (85 mg, 1.2 mmol) in dry MeOH (1.5 mL),
pre-cooled down over dry ice, was added slowly with sodium
methoxide (0.7 mL, 3.0 mmol). The resulting mixture was stirred at
-20.degree. C. for 1 hour before it was warmed up to the room temp.
LC-MS showed the reaction completed after 2 hours. After workup,
the mixture was purified by RPHPLC to afford 12a as white solid (25
mg, 49% calcd as TFA salt) after lyophilisation of the HPLC
fractions. LC-MS m/z 441.1 ([M+H]). HPLC purity (254 nm):
94.7%.
[0302] Preparation of freebase of 12a. The TFA salt was dissolved
in acetonitrile and water, and then basified using saturated
aqueous NaHCO.sub.3 to pH around 8. After removal of acetonitrile
under reduced pressure, the aqueous solution was extracted with
ethyl acetate (.times.3). The combined organic layers was dried and
evaporated to afford crude freebase which was further purified by
recrystallization in MeOH. LC-MS m/z 441.2 ([M+H].sup.+). HPLC
purity (254 nm): 99.8%. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.44
(s, 1H), 8.72 (s, 1H), 8.38 (d, J=2.4 Hz, 1H), 8.36 (d, J=8.0 Hz,
1H), 8.36 (s, 1H), 7.46 (t, J=8.0 Hz, 1H), 7.10 (dd, J=7.8, 2.2 Hz,
1H), 4.75 (septet, J=6.6 Hz, 1H), 4.05 (t, J=6.2 Hz, 2H), 3.81 (t,
J=4.8 Hz, 4H), 3.74 (t, J=4.8 Hz, 4H), 2.18 (t, J=7.0 Hz, 2H), 1.99
(quintet, J=7.0 Hz, 2H), 1.55 (d, J=6.8 Hz, 6H). .sup.13C NMR
(DMSO-d.sub.6) .delta. 169.1, 159.0, 158.4, 154.3, 152.9, 142.1,
137.9, 130.0, 125.2, 121.9, 116.9, 115.8, 67.8, 66.5, 45.7, 45.2,
29.3, 25.3, 22.3. HRMS (ESI) m/z [M+H].sup.+ calcd for
C.sub.22H2.sub.8N.sub.6O.sub.4, 441.2245; found, 441.2256.
Example 4: Synthesis of
N-hydroxy-7-(2-(3-(hydroxymethyl)phenyl)-6-morpholino-9H-purin-9-yl)hepta-
namide (FIG. 4, Compound 19f)
[0303] The reaction scheme for the synthesis of
N-hydroxy-7-(2-(3-(hydroxymethyl)phenyl)-6-morpholino-9H-purin-9-yl)hepta-
namide is shown in FIG. 9.
Step 1: Synthesis of ethyl 7-(2,6-dichloro-9H-purin-9-yl)heptanoate
(3d)
[0304] To a pre-stirred of solution of 2,6-dichloro-9H-purine 1a
(376 mg, 2.0 mmol), ethyl 7-bromoheptanoate (521 mg, 2.2 mmol) in
DMF (15 mL), was added anhydrous potassium carbonate (552 mg, 4.0
mmol) and NaI (64 mg, 0.4 mmol). The resulting mixture was stirred
at 40.degree. C. for 12 hours. LC-MS showed the reaction completed.
After workup, the crude was purified by flash chromatography
(silica, ethyl acetate/hexanes from 1:3 to 1:2) to afford 3d (480
mg, 69%).
Step 2: Synthesis of ethyl
7-(2-chloro-6-morpholino-9H-purin-9-yl)heptanoate (14b)
[0305] To a pre-stirred of solution of 3d (344 mg, 1.0 mmol) in
dioxane (10 mL), were added with morpholine (435 mg, 5.0 mmol). The
resulting mixture was stirred at 60.degree. C. for 3 hour. LC-MS
showed the reaction completed. After simple workup, crude product
of 14b (404 mg, 100%) was obtained and used for the next step of
reaction without further purification. LC-MS m/z 397.2 ([M+H]+).
.sup.1HNMR (CDCl.sub.3) .delta. 7.70 (s, 1H), 4.00-5.60 (m, 8H),
3.82 (t, J=4.6 Hz, 4H), 2.27 (t, J=7.2 Hz, 2H), 1.85 (m, 2H), 1.60
(quintet, J=7.0 Hz, 2H), 1.34 (m, 4H), 1.24 (t, J=7.0 Hz, 3H).
.sup.13C NMR (CDCl.sub.3) .delta. 173.6, 153.94, 153.93, 152.2,
118.6, 66.9, 60.3, 45.7 (br), 43.8, 34.1, 29.8, 28.5, 26.3, 24.7,
14.3.
Step 3: Synthesis of ethyl
7-(2-(3-(hydroxymethyl)phenyl)-6-morpholino-9H-purin-9-yl)heptanoate
(18f)
[0306] To a pre-stirred of solution of crude 14b (100 mg, 0.254
mmol), boronic acid 4c (76 mg, 0.5 mmol) in dioxane (5.0 mL), was
added with potassium carbonate (86 mg, 0.62 mmol) and
Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2 (10 mg). The resulting mixture
was stirred at 150.degree. C. for 1 hour under the microwave
irradiation. LC-MS showed the reaction completed after 1 hour.
After removing the solvent, the crude was suspended in DCM and
purified by flash chromatography (silica, 1% to 2% MeOH in DCM) to
afford 18f (30 mg, 52.6%). LC-MS m/z 468.3 ([M+H]+). .sup.1HNMR
(DMSO-d.sub.6) .delta. 8.34 (s, 1H), 8.27 (dt-like, J=7.2 Hz, 1H),
8.22 (s, 1H), 7.44-7.37 (m, 2H), 5.28 (t, J=5.0 Hz, 1H), 4.58 (d,
J=4.4 Hz, 2H), 4.30 (br s, 4H), 4.23 (t, J=6.8 Hz, 2H), 4.01 (q,
J=7.2 Hz, 2H), 3.77 (t, J=4.8 Hz, 4H), 2.24 (t, J=7.2 Hz, 2H), 1.86
(quintet, J=7.2 Hz, 2H), 1.49 (quintet, J=7.4 Hz, 2H), 1.22-1.38
(m, 4H), 1.14 (t, J=7.0 Hz, 3H). .sup.13C NMR (DMSO-d.sub.6)
.delta. 173.3, 157.3, 153.5, 152.3, 142.9, 141.1, 138.6, 128.4,
128.3, 126.7, 126.2, 118.7, 66.7, 63.5, 60.1, 45.6, 43.2, 33.8,
29.5, 28.2, 26.1, 24.7, 14.5.
Step 4: Synthesis of
N-hydroxy-7-(2-(3-(hydroxymethyl)phenyl)-6-morpholino-9H-purin-9-yl)hepta-
namide (19f)
[0307] To a pre-stirred of solution of 18f (30 mg, 0.064 mmol),
hydroxylamine hydrochloride (67 mg, 0.96 mmol) in dry MeOH (1.0
mL), pre-cooled down over dry ice, was added slowly with sodium
methoxide (0.37 mL, 1.6 mmol). The resulting mixture was stirred at
-20.degree. C. for 1 hour before it was warmed up to the room
temperature. LC-MS showed the reaction completed after 2 hours.
After simple workup, the mixture was purified by RPHPLC to afford
N-hydroxy-7-(2-(3-(hydroxymethyl)phenyl)-6-morpholino-9H-purin-9-yl)hepta-
namide 19f as white solid (8 mg, 22% as calcd as TFA salt). The TFA
salt was dissolved in acetonitrile and water, and then basified
using saturated aqueous NaHCO3 to pH around 8. After removal of
acetonitrile under reduced pressure, the aqueous solution was
extracted with ethyl acetate (.times.3). The combined organic
layers was dried and evaporated to afford crude freebase which was
further purified by recrystallization in MeOH. Freebase of 19f:
LC-MS m/z 455.1 ([M+H].sup.+). HPLC purity (254 nm): 97.4%. .sup.1H
NMR (DMSO-d.sub.6) .delta. 10.33 (s, 1H), 8.66 (s, 1H), 8.35 (s,
1H), 8.27 (dt-like, J=7.6 Hz, 1H), 8.23 (s, 1H), 7.44 (t, J=7.6 Hz,
1H), 7.40 (dt-like, J=7.6 Hz, 1H), 5.29 (t, J=7.6 Hz, 1H), 4.59 (d,
J=5.6 Hz, 2H), 4.31 (m, 4H), 4.23 (t, J=7.0 Hz, 2H), 3.78 (t, 4H),
1.93 (t, J=7.4 Hz, 2H), 1.87 (quintet, 2H), 1.48 (quintet, 2H),
1.29 (m, 4H); .sup.13C NMR (DMSO-d.sub.6) .delta. 169.0, 156.9,
153.0, 151.8, 142.5, 140.6, 138.2, 128.0, 127.9, 126.2, 125.8,
118.2, 66.3, 63.0, 45.1, 42.8, 32.2, 29.1, 28.0, 25.7, 25.0. HRMS
(ESI) m/z [M+H].sup.+ calcd for C.sub.23H.sub.30N.sub.6O.sub.4,
455.2402; found, 455.2405.
Example 5: Synthesis of
7-(6-(2-aminopyrimidin-5-yl)-2-morpholino-9H-purin-9-yl)-N-hydroxyheptana-
mide (FIG. 3, Compound 8a)
[0308] The reaction scheme for the synthesis of
7-(6-(2-aminopyrimidin-5-yl)-2-morpholino-9H-purin-9-yl)-N-hydroxyheptana-
mide is shown in FIG. 10.
Step 1: Synthesis of ethyl
7-(6-(2-aminopyrimidin-5-yl)-2-chloro-9H-purin-9-yl)heptanoate
(5d)
[0309] To a pre-stirred of solution of 3d (from Example 2, step 1)
(344 mg, 1.0 mmol),
5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine 4d
(240 mg, 1.1 mmol) in dioxane (15 mL), were added a solution of
K.sub.2CO.sub.3 (345 mg, 2.5 mmol) in DI water (2.0 mL). The
mixture was degassed for 30 min before it were added
Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2 (41 mg, 0.05 equiv). The
resulting mixture was heated at 82.degree. C. for 6 hours. LC-MS
showed the reaction completed. After workup, the crude was purified
by flash chromatography (silica, 33% to 50% to 100% of ethyl
acetate in hexanes) to afford 5d (150 mg, 37%).
Step 2. Synthesis of ethyl
7-(6-(2-aminopyrimidin-5-yl)-2-morpholino-9H-purin-9-yl)heptanoate
(7b)
[0310] To a pre-stirred of solution of 5d (150 mg, 0.37 mmol) in
DMF (5 mL), was added with morpholine (0.70 mL, 3.0 mmol). The
resulting mixture was heated at 80.degree. C. for 12 hours. LC-MS
showed the reaction completed. After workup, 7b (120 mg, 72%) was
obtained by recrystallization of the crude in 10% MeOH in DCM.
Step 3: Synthesis of
7-(6-(2-aminopyrimidin-5-yl)-2-morpholino-9H-purin-9-yl)-N-hydroxyheptana-
mide (8a)
[0311] To a pre-stirred of solution of 7b (70 mg, 0.155 mmol),
hydroxylamine hydrochloride (108 mg, 15.5 mmol) in dry MeOH (1.5
mL), pre-cooled down over dry ice, was added slowly with sodium
methoxide (901 uL, 3.9 mmol). The resulting mixture was stirred at
-20.degree. C. for 1 hour before it was warmed up to the room temp.
LC-MS showed the reaction completed after 3 hours. After workup,
the crude was purified by RPHPLC to afford
7-(6-(2-aminopyrimidin-5-yl)-2-morpholino-9H-purin-9-yl)-N-hydr-
oxyheptanamide 8a (15 mg, 21% calcd as TFA salt). LC-MS m/z 442.1
([M+H].sup.+). HPLC purity (254 nm): 96.6%. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.34 (s, 1H), 9.54 (s, 2H), 8.25 (s, 1H),
7.45 (s, 2H), 4.12 (t, J=6.8 Hz, 2H), 3.78 (m, 4H), 3.72 (m, 4H),
1.93 (t, J=7.2 Hz, 2H), 1.85 (m, 2H), 1.48 (m, 2H), 1.26 (m, 4H).
HRMS (ESI) m/z [M+H].sup.+ calcd for
C.sub.20H.sub.27N.sub.9O.sub.3, 442.2310; found, 442.2308.
Example 6: Synthesis of
N.sup.1-hydroxy-N.sup.8-(3-(9-isopropyl-2-morpholino-9H-purin-6-yl)phenyl-
)octanediamide (FIG. 3, Compound 12f)
[0312] The reaction scheme for the synthesis of
N.sup.1-hydroxy-N.sup.5-(3-(9-isopropyl-2-morpholino-9H-purin-6-yl)phenyl-
)octanediamide is shown in FIG. 11.
Step 1: Synthesis of methyl
8-oxo-8-((3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)amino)oct-
anoate (Scheme 5, 4b)
[0313] To a pre-stirred of solution of (3-aminophenyl)boronic ester
(164 mg, 0.75 mmol), monomethyl suberate (155 mg, 0.83 mmol),
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI)
(159 mg, 0.83 mmol) in dry DCM (15 mL), were added
hydroxybenzotriazole (HOBT, 112 mg). The mixture was stirred at
room temp for 4 hours. LC-MS showed the reaction completed. After
workup, the crude was purified by flash chromatography (silica, 20%
to 25% ethyl acetate in hexanes) to afford the product 4b (149 mg,
46%).
Step 2: Synthesis of methyl
8-((3-(2-chloro-9-isopropyl-9H-purin-6-yl)phenyl)amino)-8-oxooctanoate
(5c)
[0314] To a pre-stirred of solution of
2,6-dichloro-9-isopropyl-9H-purine 3a (59 mg, 0.26 mmol), 4b (100
mg, 0.26 mmol) in dioxane (10 mL), were added a solution of
K.sub.2CO.sub.3 (88 mg, 0.64 mmol) in DI water (1.0 mL). The
mixture was degassed for 30 min before it were added
Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2 (10 mg, 0.05 equiv). The
resulting mixture was heated at 82.degree. C. for 6 hours. LC-MS
showed the reaction completed. After workup, the crude was purified
by flash chromatography (silica, 25% to 33% of ethyl acetate in
hexanes) to afford 5c (100 mg, 85%).
Step 3: Synthesis of methyl
8-((3-(9-isopropyl-2-morpholino-9H-purin-6-yl)phenyl)amino)-8-oxooctanoat-
e (7c)
[0315] To a pre-stirred of solution of 5c (75 mg, 0.16 mmol) in DMF
(2 mL), was added morpholine (140 mg, 1.6 mmol). The resulting
mixture was heated at 80.degree. C. for 16 hours. After workup, the
crude was purified by flash chromatography (silica, 33% to 50%
ethyl acetate in hexanes) to afford 7c (46 mg, 55%).
Step 4: Synthesis of
N.sup.1-hydroxy-N.sup.5-(3-(9-isopropyl-2-morpholino-9H-purin-6-yl)phenyl-
)octanediamide (12f)
[0316] To a pre-stirred of solution of 7c (46 mg, 0.09 mmol),
hydroxylamine hydrochloride (70 mg, 0.9 mmol) in dry MeOH (1 mL),
pre-cooled down over dry ice, was added slowly with sodium
methoxide (520 .mu.L, 2.2 mmol). The resulting mixture was stirred
at -20.degree. C. for 1 hour before it was warmed up to the room
temperature. LC-MS showed the reaction completed after 2 hours.
After workup, the crude was purified by RPHPLC to afford 12f (20
mg, 35% calcd as TFA salt). LC-MS m/z 510.2 ([M+H].sup.+). .sup.1H
NMR (DMSO-d.sub.6) .delta. 10.34 (s, 1H), 10.07 (s, 1H), 8.75 (s,
1H), 8.51 (d, J=8.0 Hz, 1H), 8.37 (s, 1H), 7.92 (d, =8.8 Hz, 1H),
7.45 (t, =8.0 Hz, 1H), 4.75 (m, J=6.4 Hz, 1H), 3.81 (m, 4H), 3.74
(m, 4H), 2.34 (t, J=7.2 Hz, 2H), 1.94 (t, J=7.2 Hz, 2H), 1.61 (m,
2H), 1.54 (d, J=6.8 Hz, 6H), 1.50 (m, 2H), 1.29 (m, 4H). HPLC
purity (254 nm): 98.4%; HRMS (ESI) m/z [M+H].sup.+ calcd for
C.sub.26H.sub.35N.sub.7O.sub.4, 510.2824; found, 510.2835.
Example 7: Synthesis of
(E)-N-hydroxy-3-(4-(((2-(2-(6-methoxypyridin-3-yl)-6-morpholino-9H-purin--
9-yl)ethyl)amino)methyl)phenyl)acrylamide (FIG. 4, Compound
17a)
[0317] The reaction scheme for the synthesis of
(E)-N-hydroxy-3-(4-(((2-(2-(6-methoxypyridin-3-yl)-6-morpholino-9H-purin--
9-yl)ethyl)amino)methyl)phenyl)acrylamide is shown in FIG. 12.
Step 1: Synthesis of 4-(2-chloro-9H-purin-6-yl)morpholine (13a)
[0318] Morpholine (1.39 mL, 15.87 mmol) was added to a solution of
1a (1.0 g, 5.29 mmol) in THF (26 mL). The resulting mixture was
stirred at room temperature for 16 h. White precipitate was
observed immediately upon addition of morpholine. The white
precipitate was filtered off and washed with water (.times.2) and
methanol (.times.2) to afford 13a (1.13 g, 90%).
Step 2: Synthesis of
2-(2-chloro-6-morpholino-9H-purin-9-yl)acetonitrile (14c)
[0319] To a solution of 13a (1.18 g, 4.94 mmol) in
acetonitrile/DMSO (19:1) was added 2-iodoacetonitrile (0.71 mL,
9.87 mmol) and K.sub.2CO.sub.3 (1.36 g, 9.87 mmol). The resulting
mixture was heated at 60.degree. C. for 3 h. Then the solvents were
removed in vacuo and water was added. The aqueous layer was
extracted with DCM (.times.2) and the combined organic layers was
washed with brine (.times.1), dried over MgSO.sub.4 and evaporated
in vacuo. The crude oil was purified by flash chromatography
(silica, 50% ethyl acetate in hexanes) to afford 14c (1.31 g, 95%)
as pale brown solid.
Step 3: Synthesis of
2-(2-chloro-6-morpholino-9H-purin-9-yl)ethanamine (14d)
[0320] To a stirred solution of 14c (1.23 g, 4.42 mmol) and
NiCl.sub.2-6H.sub.2O (105 mg, 0.44 mmol) in MeOH/THF (2:1) was
added sodium borohydride (1.17 g, 30.97 mmol) in portions. The
resulting mixture was allowed to stir at room temperature for 1 h.
Then the solvents were removed in vacuo and a saturated solution of
sodium bicarbonate was added. The aqueous layer was extracted with
DCM (.times.2) and the combined organic layers was washed with
brine (.times.1), dried over MgSO.sub.4 and evaporated in vacuo.
The crude was purified by flash chromatography (silica, 10%
methanol in DCM) to afford 14d (577 mg, 46%) as colourless oil.
Step 4: Synthesis of (E)-methyl
3-(4-(((2-(2-chloro-6-morpholino-9H-purin-9-yl)ethyl)amino)methyl)phenyl)-
acrylate (15a)
[0321] To a stirred solution of 14d (576 mg, 2.04 mmol) in DCE (10
mL) was added (E)-methyl 3-(4-formylphenyl)acrylate (466 mg, 2.45
mmol), acetic acid (0.12 mL, 2.04 mmol) and sodium
triacetoxyborohydride (649 mg, 3.06 mmol) sequentially. The
resulting mixture was stirred at room temp. for 5 h. A saturated
solution of sodium bicarbonate was added to quench the reaction and
the aqueous layer was extracted with methylene chloride (.times.2).
The combined organic layers was washed with brine (.times.1), dried
over MgSO.sub.4 and evaporated in vacuo. The crude was purified by
flash chromatography (silica, 4% methanol in DCM) to 15a (414 mg,
44%) as off-white solid.
Steps 5 and 6
[0322] by following analogous procedures of Example 4, steps 3 and
4, the title compound
(E)-N-hydroxy-3-(4-(((2-(2-(6-methoxypyridin-3-yl)-6-morpholino-9H-purin--
9-yl)ethyl)amino)methyl)phenyl)acrylamide (17a) was obtained as TFA
salt. LC-MS m/z 531 ([M+H].sup.+). .sup.1HNMR (DMSO-d.sub.6)
.delta. 10.82 (br s, 1H), 9.16 (d, J=2.4 Hz, 1H), 9.07 (br s, 2H),
8.54 (dd, J=2.4 Hz, 8.8 Hz, 1H), 8.20 (s, 1H), 7.56 (d, J=8.0 Hz,
2H), 7.46 (d, J=8.0 Hz, 2H), 7.42 (overlapping, 1H), 6.88 (d, J=8.8
Hz, 1H), 6.50 (d, J=16 Hz, 1H), 4.60 (t, J=5.2 Hz, 2H), 4.34-4.28
(m, 6H), 3.92 (s, 3H), 3.77 (t, J=4.8 Hz, 4H), 3.60 (t-like, 2H).
HRMS (ESI) m/z [M+H].sup.+ calcd for
C.sub.27H.sub.31N.sub.8O.sub.4, 531.2463; found, 531.2473.
Example 8: Synthesis of
6-((6-(2-aminopyrimidin-5-yl)-9-isopropyl-2-morpholino-9H-purin-8-yl)amin-
o)-N-hydroxyhexanamide (FIG. 5, Compound 24a)
[0323] The reaction scheme for the synthesis of
6-((6-(2-aminopyrimidin-5-yl)-9-isopropyl-2-morpholino-9H-purin-8-yl)amin-
o)-N-hydroxyhexanamide is shown in FIG. 13.
Step 1: Synthesis of
5-(8-bromo-9-isopropyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine
(22a)
[0324] To a pre-stirred of solution of
5-(9-isopropyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine 7d (400
mg, 1.13 mmol) in CHCl.sub.3 (15 mL) was added slowly with NBS (362
mg, 2.03 mmol) in ice-water bath. The resulting mixture was warmed
up to room temperature for 2 hours. After workup, the crude was
purified by flash chromatography (silica, 25% to 33% of ethyl
acetate in DCM) to afford 22a (225 mg, 44%).
Step 2: Synthesis of methyl
6-((6-(2-aminopyrimidin-5-yl)-9-isopropyl-2-morpholino-9H-purin-8-yl)amin-
o)hexanoate (23a)
[0325] To a pre-stirred solution of 22a (52 mg, 0.12 mmol) in NMP
(1 mL), was added with methyl 6-amino hexanoate hydrochloride (380
mg, 1.88 mmol). The resulting mixture was heated at 130.degree. C.
for 12 hours. After workup, the crude was purified by flash
chromatography (silica, 20% to 80% ethyl acetate in hexanes) to
afford 23a (44 mg, 73%).
Step 3: Synthesis of
6-((6-(2-aminopyrimidin-5-yl)-9-isopropyl-2-morpholino-9H-purin-8-yl)amin-
o)-N-hydroxyhexanamide (24a)
[0326] To a pre-stirred solution of 23a (35 mg, 0.072 mmol),
hydroxylamine hydrochloride (51 mg, 0.72 mmol) in dry MeOH (1.0
mL), pre-cooled down over dry ice, was added slowly with sodium
methoxide (334 .mu.L, 1.44 mmol). The resulting mixture was stirred
at -20.degree. C. for 1 hour before it was warmed up to the room
temperature. LC-MS showed the reaction completed after 2 hours.
After workup, the crude was purified by RPHPLC to afford 24a (30
mg, 69% calcd with TFA salt). LC-MS m/z 485.3 ([M+H]). .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.40 (s, 1H), 9.21 (s, 2H), 7.78 (s, 1H),
7.41 (s, 2H), 4.65 (m, J=6.8 Hz, 1H), 3.43 (m, 4H), 1.98 (m, 2H),
1.65 (m, 2H), 1.58 (d, J=6.8 Hz, 6H), 1.33 (m, 2H). HPLC purity
(254 nm): 99.2%. HRMS (ESI) m/z [M+H].sup.+ calcd for
C.sub.22H.sub.32N.sub.10O.sub.3, 485.2732; found, 485.2749.
Example 9: Synthesis of
4-((((2-(2-aminopyrimidin-5-yl)-9-ethyl-6-morpholino-9H-purin-8-yl)methyl-
)(methyl)amino)methyl)-N-hydroxybenzamide (FIG. 6, Compound
35h)
[0327] The reaction scheme for the synthesis of
4-((((2-(2-aminopyrimidin-5-yl)-9-ethyl-6-morpholino-9H-purin-8-yl)methyl-
)(methyl)amino)methyl)-N-hydroxybenzamide is shown in FIG. 14.
Step 1: Synthesis of
2-chloro-9-ethyl-6-morpholino-9H-purine-8-carbaldehyde (29a)
[0328] To a solution of
4-(2-chloro-9-ethyl-9H-purin-6-yl)morpholine 14a (600 mg, 2.25
mmol) in THF (11 mL) was added a solution of n-BuLi in hexanes (2.5
M, 1.08 ml, 2.7 mmol) at -78.degree. C. The resulting mixture was
stirred at -78.degree. C. for 1 h and then DMF (0.26 mL, 3.37 mmol)
was added dropwise over a period of 10 min. Then the reaction
mixture was allowed to stir at room temperature for additional 2 h
before quenching the reaction mixture with ice. The aqueous layer
was extracted with ethyl acetate (.times.2) and washed with brine
(.times.1), dried over MgSO.sub.4, filtered and concentrated in
vacuo to yield crude yellow oil. The crude was purified by flash
chromatography eluting with hexanes/ethyl acetate (7:3) to afford
29a (398 mg, 60%) as off-white solid. LC-MS m/z 296 ([M+H].sup.+).
.sup.1HNMR (CDCl.sub.3) .delta. 9.88 (s, 1H), 4.70 (br s, 2H), 4.59
(q, J=7.1 Hz, 2H), 4.06 (br s, 2H), 3.86 (s, 4H), 1.41 (t, J=7.1
Hz, 3H).
Step 2: Synthesis of
1-(2-chloro-9-ethyl-6-morpholino-9H-purin-8-yl)-N-methylmethanamine
(30a)
[0329] To a solution of
2-chloro-9-ethyl-6-morpholino-9H-purine-8-carbaldehyde 29a (300 mg,
1.02 mmol) in DCE/MeOH (2:1) was added a solution of methylamine in
methanol (9.8 M, 0.83 mL, 8.14 mmol). White precipitate was
observed after 30 min of stirring at room temperature. The
resulting mixture was then stirred for additional 5 h and the
solvent was removed in vacuo to afford off-white solid. Then the
crude solid was dissolved in a solution of DCM/MeOH (4:1) and
sodium borohydride (115 mg, 3.05 mmol) was added in portions. The
resulting mixture was stirred at room temperature for 15 h. The
reaction mixture was evaporated in vacuo and water was added. The
aqueous layer was extracted with methylene chloride (.times.2),
washed with brine (.times.1), dried over MgSO.sub.4, filtered and
concentrated in vacuo to yield crude off-white solid. The crude was
purified by flash chromatography eluting with methylene
chloride/methanol (24:1) to afford 30a (256 mg, 82%) as white
solid.
Step 3A: Synthesis of (E)-methyl
3-(4-((((2-chloro-9-ethyl-6-morpholino-9H-purin-8-yl)methyl)(methyl)amino-
)methyl)phenyl)acrylate (33a)
[0330] To a stirred solution of 30a (242 mg, 0.78 mmol) in DCE (4.0
mL) was added (E)-methyl 3-(4-formylphenyl)acrylate (163 mg, 0.86
mmol), acetic acid (47 .mu.L, 0.78 mmol) and sodium
triacetoxyborohydride (248 mg, 1.17 mmol) sequentially. The
resulting mixture was stirred at room temperature for 5 h. A
saturated solution of sodium bicarbonate was added to quench the
reaction and the aqueous layer was extracted with methylene
chloride (.times.2). The combined organic layers was washed with
brine (.times.1), dried with MgSO.sub.4 and evaporated in vacuo.
The crude was purified by flash chromatography eluting with
hexanes/ethyl acetate (3:2) to afford 33a (328 mg, 87%) as white
solid. LC-MS m/z 485 ([M+H].sup.+). .sup.1HNMR (CDCl.sub.3) .delta.
7.68 (d, J=16 Hz, 1H), 7.49 (d, J=8.0 Hz, 2H), 7.33 (d, J=8.0 Hz,
2H), 6.43 (d, J=16 Hz, 1H), 4.27 (q, J=7.2 Hz, 2H), 4.27 (masked
peak, 4H), 3.81 (s, 3H), 3.83-3.80 (masked peak, 4H), 3.70 (s, 2H),
3.59 (s, 2H), 2.23 (s, 3H), 1.34 (t, J=7.2 Hz, 3H).
Step 3B: Synthesis of methyl
4-((((2-chloro-9-ethyl-6-morpholino-9H-purin-8-yl)methyl)(methyl)amino)me-
thyl)benzoate (33b)
[0331] To a solution of
1-(2-chloro-9-ethyl-6-morpholino-9H-purin-8-yl)-N-methylmethanamine
30a (485 mg, 1.57 mmol) in THF (7.8 mL) was added methyl
4-(bromomethyl)benzoate (466 mg, 2.03 mmol) and triethylamine (0.28
mL, 2.03 mmol). The resulting mixture was stirred at room
temperature for 16 h and the solvent was removed in vacuo. Then
saturated sodium bicarbonate was added and the aqueous layer was
extracted with ethyl acetate (.times.2) and washed with brine
(.times.1), dried over MgSO.sub.4, filtered and concentrated in
vacuo to yield crude yellow oil. The crude was purified by flash
chromatography eluting with hexanes/ethyl acetate (3:2) to afford
33b (703 mg, 98%) as colourless oil.
Step 4: Synthesis of methyl
4-((((2-(2-aminopyrimidin-5-yl)-9-ethyl-6-morpholino-9H-purin-8-yl)methyl-
)(methyl)amino)methyl)benzoate (34 h)
[0332] To a solution of 33b (203 mg, 0.44 mmol) in dioxane (0.9 mL)
was added an aqueous solution of K.sub.2CO.sub.3 (122 mg, 0.89
mmol) followed by
5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine 4a
(147 mg, 0.66 mmol) and Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2 (18 mg,
0.02 mmol). The reaction mixture was then heated up in a microwave
reactor at 150.degree. C. for a period of 20 min. Dioxane was
removed and the aqueous layer was extracted with ethyl acetate
(.times.3). The combined organic extracts were dried with
MgSO.sub.4 and evaporated in vacuo. The brown residue was purified
by flash chromatography eluting with hexanes/ethyl acetate (2:3) to
DCM/MeOH (24:1) to afford 34 h (168 mg, 70%) as a pale brown solid.
LC-MS m/z 518 ([M+H]). .sup.1HNMR (CDCl.sub.3) .delta. 9.26 (s,
2H), 7.99 (d, J=8.0 Hz, 2H), 7.40 (d, J=8.0 Hz, 2H), 5.25 (s, 2H),
4.37-4.32 (m, 6H), 3.91 (s, 3H), 3.86 (t, J=4.8 Hz, 4H), 3.75 (s,
2H), 3.65 (s, 2H), 2.25 (s, 3H), 1.41 (t, J=7.2 Hz, 3H).
Step 5: Synthesis of
4-((((2-(2-aminopyrimidin-5-yl)-9-ethyl-6-morpholino-9H-purin-8-yl)methyl-
)(methyl)amino)methyl)-N-hydroxybenzamide (35 h)
[0333] NaOMe (1.23 mL, 5.36 mmol) was added dropwise to a solution
of 34 h (79 mg, 0.15 mmol) and hydroxylamine hydrochloride (106 mg,
1.53 mmol) in DCM/MeOH (4.2 mL, 2:3 v/v) at -78.degree. C. The
reaction mixture was then warmed to room temp and stirred for 30
min. Then the reaction mixture was diluted with water to obtain a
clear solution and neutralised with 6N HCl. The crude mixture was
purified by RPHPLC to provide the title compound 35h (60 mg, 53% as
bis-TFA salt). Alternatively, the solvent of the RPHPLC fractions
was removed in vacuo and saturated solution of sodium bicarbonate
was added into purified compound and extracted with ethyl acetate
(.times.3). The combined organic layer was washed with brine
(.times.1), dried over MgSO.sub.4, and concentrated in vacuo to
deliver 35 h (17 mg, 22% as freebase) as white solid. TFA salt of
35 h: LC-MS m/z 519 ([M+H].sup.+). .sup.1HNMR (DMSO-d.sub.6)
.delta. 11.32 (br s, 1H), 9.14 (s, 2H), 7.84 (d, J=8.0 Hz, 2H),
7.58 (d, J=7.6 Hz, 2H), 7.20 (br s, 2H), 4.63 (br s, 2H), 4.30-4.27
(m, 8H), 3.79 (t, J=4.4 Hz, 4H), 2.78 (br s, 3H), 1.35 (t, J=7.2
Hz, 3H). Freebase of 35 h: LC-MS m/z 519 ([M+H]f). .sup.1HNMR
(DMSO-d.sub.6) .delta. 11.20 (s, 1H), 9.10 (s, 2H), 9.03 (s, 1H),
7.73 (d, J=8.4 Hz, 2H), 7.41 (d, J=8.0 Hz, 2H), 7.07 (s, 2H),
4.34-4.25 (m, 6H), 3.79 (s, 2H), 3.76-3.74 (m, 4H), 3.64 (s, 2H),
2.11 (s, 3H), 1.36 (t, J=6.8 Hz, 3H). HRMS (ESI) m/z [M+H].sup.+
calcd for C.sub.25H.sub.31N.sub.10O.sub.3, 519.2575; found,
519.2593.
[0334] The following compounds in Table 1 were made by using
synthetic routes described in Schemes 1-5 and procedures analogous
to those in Examples 3-9.
TABLE-US-00004 TABLE 1 Table showing selected compounds Compound
Chemical Structure Chemical Name and Analytical Data 5b
##STR00030## 4-(9-isopropyl-2-morpholino-9H-purin-6- yl)phenol.
LC-MS m/z 340 ([M + H].sup.+). .sup.1HNMR (CDCl.sub.3) .delta. 8.56
(d, J = 8.8 Hz, 1H), 7.88 (s, 1H), 6.92 (d, J = 8.4 Hz, 1H), 4.80
(septet, J = 6.8 Hz, 1H), 3.95-3.85 (m, 8H), 1.61 (d, J = 6.8 Hz,
6H). 5c ##STR00031## (4-(9-isopropyl-2-morpholino-9H-purin-6-
yl)phenyl)methanol. LC-MS m/z 354 ([M + H].sup.+). .sup.1HNMR
(CDCl.sub.3) .delta. 8.72 (d, J = 8.4 Hz, 2H), 7.86 (s, 1H), 7.50
(d, J = 8.0 Hz, 2H), 4.82-4.76 (m, 3H), 3.95-3.84 (m, 8H), 1.89 (t,
J = 6.0 Hz, 1H), 1.61 (d, J = 6.8 Hz, 6H). 5d ##STR00032##
[4-(2-Chloro-9-isopropyl-9H-purin-6-yl)- phenyl]-methanol. LC-MS
m/z 303; 305 ([M + H].sup.+). .sup.1HNMR (CDCl.sub.3) .delta. 8.79
(d, J = 8.4 Hz, 2H), 8.17 (s, 1H), 7.55 (d, J = 8.6 Hz, 2H),
5.02-4.93 (m, 1H), 4.80 (d, J = 5.9 Hz, 2H), 1.89 (t, J = 6.0 Hz,
1H), 1.64 (d, J = 6.8 Hz, 6H). 5e ##STR00033##
(3-(2-chloro-9-isopropyl-9H-purin-6- yl)phenyl)methanol. LC-MS m/z
303; 305 ([M + H].sup.+). .sup.1HNMR (CDCl.sub.3) .delta. 8.73-8.70
(masked peak, 2H), 8.16 (s, 1H), 7.57-7.55 (masked peak, 2H),
5.00-4.94 (m, 1H), 4.83 (s, 2H), 1.66 (d, J = 6.8 Hz, 6H). 5f
##STR00034## [3-(9-Isopropyl-2-morpholin-4-yl-9H-
purin-6-yl)-phenyl]-methanol. LC-MS m/z 354 ([M + H].sup.+).
.sup.1HNMR (CDCl.sub.3) .delta. 8.68 (s, 1H), 8.61 (dt, J = 6.8,
2.0 Hz, 1H), 7.82 (s, 1H), 7.54-7.50 (m, 2H), 4.81 (s, 2H), 4.78
(masked peak, 1H), 3.94-3.83 (m, 8H), 1.59 (d, J = 6.8 Hz, 6H). 11i
##STR00035## Methyl 5-(4-(9-isopropyl-2-morpholino-
9H-purin-6-yl)phenoxy)pentanoate. LC-MS m/z 454 ([M + H].sup.+).
.sup.1HNMR (CDCl.sub.3) .delta. 8.72 (dd, J = 9.2, 2.0 Hz, 2H),
7.84 (s, 1H), 7.01 (dd, J = 9.2, 2.0 Hz, 2H), 4.81-4.73 (m, 1H),
4.06 (t, J = 5.6 Hz, 2H), 3.94-3.83 (m, 8H), 3.68 (s, 3H), 2.42 (t,
J = 7.0 Hz, 2H), 1.88-1.85 (m, 4H), 1.60 (d, J = 6.4 Hz, 6H). 11h
##STR00036## Ethyl 4-(4-(9-isopropyl-2-morpholino-9H-
purin-6-yl)phenoxy)butanoate. LC-MS m/z 454 ([M + H].sup.+).
.sup.1HNMR (CDCl.sub.3) .delta. 8.72 (dd, J = 9.2, 2.0 Hz, 2H),
7.84 (s, 1H), 7.01 (dd, J = 8.8, 2.0 Hz, 2H), 4.81-4.75 (m, 1H),
4.15 (q, J = 7.2 Hz, 2H), 4.09 (t, J = 6.0 Hz, 2H), 3.94-3.83 (m,
8H), 2.54 (t, J = 7.2 Hz, 2H), 2.18-2.12 (m, 2H), 1.60 (d, J = 6.8
Hz, 6H), 1.27 (t, J = 7.2 Hz, 3H). 11j ##STR00037## Ethyl
6-(4-(9-isopropyl-2-morpholino-9H- purin-6-yl)phenoxy)hexanoate.
LC-MS m/z 482 ([M + H].sup.+). .sup.1HNMR (CDCl.sub.3) .delta. 8.72
(dd, J = 9.2, 2.0 Hz, 2H), 7.84 (s, 1H), 7.01 (dd, J = 8.8, 2.0 Hz,
2H), 4.81-4.75 (m, 1H), 4.13 (q, J = 7.2 Hz, 2H), 4.04 (t, J = 6.4
Hz, 2H), 3.94-3.83 (m, 8H), 2.35 (t, J = 7.2 Hz, 2H), 1.88-1.81 (m,
2H), 1.76-1.68 (m, 2H), 1.60 (d, J = Hz, 6 H) 1.57-1.53 (m, 2H),
1.26 (t, J = 7.2 Hz, 3H). 11k ##STR00038## Ethyl
7-(4-(9-isopropyl-2-morpholino-9H- purin-6-yl)phenoxy)heptanoate.
LC-MS m/z 496 ([M + H].sup.+). .sup.1HNMR (CDCl3) .delta. 8.72 (dd,
J = 8.8, 2.0 Hz, 2H), 7.84 (s, 1H), 7.01 (dd, J = 8.8, 2.0 Hz, 2H),
4.81-4.75 (m, 1H), 4.13 (q, J = 7.2 Hz, 2H), 4.04 (t, J = 6.4 Hz,
2H), 3.94-3.83 (m, 8H), 2.32 (t, J = 7.4 Hz, 2H), 1.86-1.79 (m,
2H), 1.71- 1.64 (m, 2H), 1.60 (d, J = 6.8 Hz, 6H), 1.55-1.45 (m,
4H), 1.26 (t, J = 7.2 Hz, 3H). 11l ##STR00039## Methyl
5-((4-(9-isopropyl-2-morpholino-
9H-purin-6-yl)benzyl)oxy)pentanoate. LC- MS m/z 468 ([M +
H].sup.+). .sup.1HNMR (CDCl3) .delta. 8.70 (d, J = 8.4 Hz, 2H),
7.86 (s, 1H), 7.48 (d, J = 8.0 Hz, 2H), 4.82-4.76 (m, 1H), 4.58 (s,
2H), 3.95-3.84 (m, 8H), 3.68 (s, 3H), 3.50 (t, J = 6.0 Hz, 2H),
2.35 (t, J = 7.2 Hz, 2H), 1.77-1.60 (m, 10H). 11m ##STR00040##
6-[4-(9-Isopropyl-2-morpholin-4-yl-9H-
purin-6-yl)-benzyloxy]-hexanoic acid ethyl ester. LC-MS m/z 496 ([M
+ H].sup.+). .sup.1HNMR (CDCl.sub.3) .delta. 8.74 (d, J = 8.4 Hz, 2
H), 7.90 (s, 1H), 7.51 (d, J = 8.4 Hz, 2 H), 4.85-4.79 (m, 1H),
4.62 (s, 2 H), 4.16 (q, J = 7.1 Hz, 2H), 3.99-3.88 (m, 8H), 3.52
(t, J = 6.6 Hz, 2H), 2.35 (t, J = 7.5 Hz, 2H), 1.71-1.65 (masked
peak, 4H), 1.65 (d, J = 6.8 Hz, 6H), 1.48-1.44 (m, 2H), 1.29 (t, J
= 7.1 Hz, 3H). 11n ##STR00041##
7-[4-(9-Isopropyl-2-morpholin-4-yl-9H-
purin-6-yl)-benzyloxy]-heptanoic acid ethyl ester. LC-MS m/z 510
([M + H].sup.+). .sup.1HNMR (CDCl3) .delta. 8.70 (d, J = 8.3 Hz,
2H), 7.86 (s, 1H), 7.48 (d, J = 8.4 Hz, 2H), 4.82-4.78 (m, 1H),
4.59 (s, 2H), 4.12 (q, J = 7.1 Hz, 2H), 3.95-3.84 (m, 8H), 3.47 (t,
J = 6.6 Hz, 2H), 2.29 (t, J = 7.5 Hz, 2H), 1.65-1.60 (masked peak,
8H), 1.40-1.34 (m, 6H), 1.25 (t, J = 7.2 Hz, 3H). 11s ##STR00042##
7-[3-(9-Isopropyl-2-morpholin-4-yl-9H-
purin-6-yl)-benzyloxy]-heptanoic acid ethyl ester. LC-MS m/z 510
([M + H].sup.+). .sup.1HNMR (CDCl.sub.3) .delta. 8.71 (dt, J = 6.8,
2.0 Hz, 1H), 8.67 (s, 1H), 7.91 (s, 1H), 7.55-7.53 (m, 2H),
4.85-4.81 (m, 1H), 4.67 (s, 2H), 4.15 (q, J = 7.1 Hz, 2H),
3.99-3.88 (m, 8H), 3.54 (t, J = 6.6 Hz, 2H), 2.32 (t, J = 7.6 Hz,
2H), 1.66 (masked peak, 10H), 1.46-1.35 (m, 4H), 1.28 (t, J = 7.1
Hz, 3H). 11w ##STR00043## Methyl 2-((4-(9-isopropyl-2-morpholino-
9H-purin-6-yl)benzyl)oxy)acetate. LC-MS m/z 426 ([M + H].sup.+).
.sup.1HNMR (CDCl.sub.3) .delta. 8.72 (d, J = 8.3 Hz, 2H), 7.87 (s,
1H), 7.51 (d, J = 8.4 Hz, 2H), 4.82-4.76 (m, 1H), 4.73 (s, 2H),
4.13 (s, 2H), 3.95-3.83 (m, 8H), 3.78 (s, 3H), 1.61 (d, J = 6.8 Hz,
6H). 11p ##STR00044## Methyl 2-((3-(9-isopropyl-2-morpholino-
9H-purin-6-yl)benzyl)oxy)acetate. LC-MS m/z 426 ([M + H].sup.+).
.sup.1HNMR (CDCl.sub.3) .delta. 8.73-8.70 (m, 1H), 8.65 (s, 1H),
7.87 (s, 1H), 7.53 (d, J = 5.2 Hz, 2H), 4.83-4.76 (masked peak,
3H), 4.15 (s, 2H), 3.95-3.84 (m, 8H), 3.77 (s, 3H), 1.61 (d, 6H).
11x ##STR00045## Methyl 2-(4-(9-isopropyl-2-morpholino-
9H-purin-6-yl)phenoxy)acetate. LC-MS m/z 412 ([M + H].sup.+).
.sup.1HNMR (CDCl.sub.3) .delta. 8.74 (dd, J = 6.9, 2.1 Hz, 2H),
7.84 (s, 1H), 7.04 (dd, J = 6.9, 2.1 Hz, 2H), 4.80-4.76 (m, 1H),
4.72 (s, 2H), 3.94-3.83 (m, 11H), 1.60 (d, J = 6.8 Hz, 6H).
##STR00046## Methyl 4-((4-(9-isopropyl-2-morpholino-
9H-purin-6-yl)phenoxy)methyl)benzoate. LC-MS m/z 488 ([M +
H].sup.+). .sup.1HNMR (CDCl.sub.3) .delta. 8.74 (d, J = 9.0 Hz,
2H), 8.06 (d, J = 8.4 Hz, 2H), 7.84 (s, 1H), 7.53 (d, J = 8.4 Hz,
2H), 7.09 (d, J = 9.0 Hz, 2H), 5.21 (s, 1H), 4.62-4.58 (m, 1H),
3.93-3.83 (m, 11H), 1.60 (d, J = 6.8 Hz, 6H). 30b ##STR00047##
Methyl (6-(((2-chloro-9-ethyl-6- morpholino-9H-purin-8-
yl)methyl)amino)hexanoate. LC-MS m/z 425 ([M + H].sup.+).
.sup.1HNMR (CDCl3) .delta. 4.34- 4.18 (m, 6H), 3.95 (s, 2H),
3.83-3.80 (m, 4H), 3.67 (s, 3H), 2.70 (t, J = 7.1 Hz, 2H), 2.32 (t,
J = 7.5 Hz, 2H), 1.69-1.63 (m, 2H), 1.59-1.51 (m, 2H), 1.44-1.34
(m, 5H). 31a ##STR00048## Methyl 6-(((2-(2-aminopyrimidin-5-yl)-9-
ethyl-6-morpholino-9H-purin-8- yl)methyl)amino)hexanoate. LC-MS m/z
484 ([M + H].sup.+). .sup.1HNMR (CDCl.sub.3) .delta. 9.27 (s, 2H),
4.34-4.27 (m 6H), 3.98 (s, 2H), 3.87- 3.85 (m 4H), 3.67 (s, 3H),
2.72 (t, J = 7.0 Hz, 2H), 2.32 (t, J = 7.4 Hz, 2H), 1.69-1.62 (m,
2H), 1.58-1.53 (m, 2H), 1.47-1.37 (m, 5H). 33a ##STR00049##
(E)-methyl 3-(4-((((2-chloro-9-ethyl- 6-morpholino-9H-purin-8-
yl)methyl)(methyl)amino)methyl)phenyl) acrylate. LC-MS m/z 485 ([M
+ H].sup.+). .sup.1HNMR (CDCl.sub.3) .delta. 7.68 (d, J = 16 Hz,
1H), 7.49 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 6.43 (d, J
= 16 Hz, 1H), 4.27 (q, J = 7.2 Hz, 2H), 4.27 (masked peak, 4H),
3.81 (s, 3H), 3.83-3.80 (masked peak, 4H), 3.70 (s, 2H), 3.59 (s,
2H), 2.23 (s, 3H), 1.34 (t, J = 7.2 Hz, 3H). 34a ##STR00050##
(E)-methyl 3-(4-((((2-(2-aminopyrimidin-5-
yl)-9-ethyl-6-morpholino-9H-purin-8-
yl)methyl)(methyl)amino)methyl)phenyl) acrylate. LC-MS m/z 544 ([M
+ H].sup.+). .sup.1HNMR (CDCl.sub.3) .delta. 9.26 (s, 2H), 7.67 (d,
J = 16 Hz, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H),
6.42 (d, J = 16 Hz, 1H), 5.28 (s, 2H), 4.38-4.32 (m, 6H), 3.86 (t,
J = 4.8 Hz, 4H), 3.81 (s, 3H), 3.74 (s, 2H), 3.61 (s, 2H), 2.25 (s,
3H), 1.40 (t, J = 7.2 Hz, 3H). 34b ##STR00051## (E)-methyl
3-(4-((((2-(4-carbamoylphenyl)- 9-ethyl-6-morpholino-9H-purin-8-
yl)methyl)(methyl)amino)methyl)phenyl) acrylate. LC-MS m/z 570 ([M
+ H].sup.+). .sup.1HNMR (CDCl.sub.3) .delta. 8.56 (d, J = 8.4 Hz,
2H), 7.92 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 16.0 Hz, 1H), 7.52 (d,
J = 8.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 6.46 (d, J = 16.0 Hz,
1H), 4.46-4.40 (m, 6H), 3.92 (t, J = 4.8 Hz, 4H), 3.84 (s, 3H),
3.80 (s, 2H), 3.66 (s, 2H), 2.30 (s, 3H), 1.47 (t, J = 7.2 Hz, 3H).
34c ##STR00052## (E)-methyl 3-(4-((((2-(3-carbamoylphenyl)-
9-ethyl-6-morpholino-9H-purin-8-
yl)methyl)(methyl)amino)methyl)phenyl) acrylate. LC-MS m/z 570 ([M
+ H].sup.+). .sup.1HNMR (CDCl.sub.3) .delta. 8.84 (t, J = 1.6 Hz,
1H), 8.62 (dt, J = 8.0, 1.2 Hz, 1H), 7.89 (dt, J = 7.6, 1.2 Hz,
1H), 7.67 (d, J = 16.0 Hz, 1H), 7.54 (t, J = 7.6 Hz, 1H), 7.47 (d,
J = 8.0 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 6.42 (d, J = 16.0 Hz,
1H), 4.42-4.37 (m, 6H), 3.88 (t, J = 4.8 Hz, 4H), 3.80 (s, 3H),
3.76 (s, 2H), 3.62 (s, 2H), 2.27 (s, 3H), 1.43 (t, J = 7.2 Hz, 3H).
34d ##STR00053## (E)-methyl 3-(4-((((2-(3-acetamidophenyl)-
9-ethyl-6-morpholino-9H-purin-8-
yl)methyl)(methyl)amino)methyl)phenyl) acrylate. LC-MS m/z 584 ([M
+ H].sup.+). .sup.1HNMR (CDCl.sub.3) .delta. 8.35 (t, like, 1H),
8.24 (d, J = 8.0 Hz, 1H), 7.87 (dd, J = 8.0, 1.6 Hz, 1H), 7.71 (d,
J = 16.0 Hz, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.45 (t, J = 8.0 Hz,
1H), 7.38 (d, J = 8.4 Hz, 2H), 7.36 (overlapped peak, 1H), 6.46 (d,
J = 16.0 Hz, 1H), 4.45-4.41 (m, 6H), 3.91 (t, J = 4.8 Hz, 4H), 3.85
(s, 3H), 3.79 (s, 2H), 3.65 (s, 2H), 2.30 (s, 3H), 2.26 (s, 3H),
1.46 (t, J = 7.2 Hz, 3H).
[0335] The following hydroxamates were made by using synthetic
routes described in Schemes 1-5 and procedures analogous to those
in Examples 3-9. The compounds have been assigned an arbitrary
identification number (denoted by EX) and the corresponding
compounds found in the schemes in Example 2 as well as their
structures and analytical data are shown below in Table 2.
TABLE-US-00005 TABLE 2 Table showing selected hydroxamate compounds
EX Compound Chemical Structure Chemical Name and Data 1 12a See
FIG. 8, compound 12a See Example 3, Step 4, compound 12a 2 19f See
FIG. 9, compound 19f See Example 4, Step 4, compound 19f 3 8a See
FIG. 10, compound 8a See Example 5, Step 3, compound 8a 4 12f See
FIG. 11, compound 12f See Example 6, Step 4, compound 12f 5 17a See
FIG. 12, compound 17a See Example 7, steps 5 and 6, compound 17a 6
24a See FIG. 13, compound 24a See Example 8, Step 3, compound 24a 7
35h See FIG. 14, compound 35h See Example 9, Step 5, compound 35h 8
12b ##STR00054## N.sup.1-hydroxy-N.sup.9-(3-(9-isopropyl-2-
morpholino-9H-purin-6- yl)phenyl)nonanediamide. LC-MS m/z 524.2 ([M
+ H].sup.+). HPLC purity (254 nm): 98.9%. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.34 (s, 1H), 10.08 (s, 1H), 8.75 (s, 1H),
8.51 (d, J = 8.0 Hz, 1H), 8.36 (s, 1H), 7.92 (d, J = 8.8 Hz, 1H),
7.45 (t, J = 8.0 Hz, 1H), 4.75 (m, J = 6.8 Hz, 1H), 3.81 (m, 4H),
3.74 (m, 4H), 2.33 (t, J = 7.6 Hz, 2H), 1.94 (t, J = 7.6 Hz, 2H),
1.61 (m, 2H), 1.55 (d, J = 7.6 Hz, 6H), 1.50 (m, 2H), 1.30 (m, 4H).
9 12c ##STR00055## N.sup.1-hydroxy-N.sup.5-(3-(9-isopropyl-2-
morpholino-9H-purin-6- yl)phenyl)glutaramide. LC-MS m/z 468.1 ([M +
H].sup.+). HPLC purity (254 nm): 94.0%. .sup.1H NMR (DMSO-d.sub.6)
.delta. 10.41 (s, 1H), 10.11 (s, 1H), 8.75 (s, 1H), 8.51 (d, J =
8.0 Hz, 1H), 8.36 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.45 (t, J =
8.0 Hz, 1H), 4.75 (m, J = 6.8 Hz, 1H), 3.81 (m, 4H), 3.74 (m, 4H),
2.36 (t, J = 7.6 Hz, 2H), 2.03 (t, J = 7.6 Hz, 2H), 1.83 (m, 2H),
1.54 (d, J = 6.8 Hz, 6H). 10 12d ##STR00056##
N-hydroxy-5-(3-(9-isopropyl-2- morpholino-9H-purin-6-
yl)phenoxy)pentanamide. LC-MS m/z 455.1 ([M + H].sup.+). HPLC
purity (254 nm): 95.1%. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.42
(s, 1H), 10.13 (s, 1H), 8.39 (s, 1H), 8.34 (s, 1H), 8.31 (d, J =
7.6 Hz, 1H), 7.45 (t, J = 8.0 Hz, 1H), 7.11 (dd, J = 8.4, 2.4 Hz,
1H), 4.75 (m, J = 6.8 Hz, 1H), 4.05 (m, 4H), 3.74 (m, 4H), 2.04 (t,
J = 7.2 Hz, 2H), 1.72 (m, 4H), 1.54 (d, J = 6.8 Hz, 6H). 11 12e
##STR00057## N-hydroxy-6-(3-(9-isopropyl-2- morpholino-9H-purin-6-
yl)phenoxy)hexanamide. LC-MS m/z 469.1 ([M + H].sup.+). HPLC purity
(254 nm): 97.0%. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.40 (s, 1H),
10.16 (s, 1H), 8.41 (s, 1H), 8.35 (s, 1H), 8.31 (d, J = 7.6 Hz,
1H), 7.45 (t, J = 8.0 Hz, 1H), 7.10 (dd, J = 8.0, 2.4 Hz, 1H), 4.75
(m, J = 6.8 Hz, 1H), 4.03 (m, 4H), 3.79 (m, 4H), 1.99 (t, J = 7.2
Hz, 2H), 1.76 (m, 4H), 1.59 (m, 2H), 1.54 (d, J = 6.8 Hz, 6H), 1.43
(m, 2H). 12 12g ##STR00058## N-hydroxy-7-(3-(9-isopropyl-2-
morpholino-9H-purin-6- yl)phenoxy)heptanamide. LC-MS m/z 483.2 ([M
+ H].sup.+). HPLC purity (254 nm): 98.8%. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.37 (s, 1H), 9.75 (s, 1H), 8.39 (s, 1H),
8.35 (s, 1H), 8.31 (d, J = 7.2 Hz, 1H), 7.45 (t, J = 8.0 Hz, 1H),
7.10 (dd, J = 8.4, 2.4 Hz, 1H), 4.75 (m, J = 6.8 Hz, 1H), 4.03 (t,
J = 6.4 Hz, 2H), 3.80 (m, 4H), 3.74 (m, 4H), 1.96 (t, J = 7.2 Hz,
2H), 1.75 (m, 2H), 1.55 (d, J = 6.8 Hz, 6H), 1.52 (m, 2H), 1.43 (m,
2H), 1.32 (m, 2H). 13 12h ##STR00059##
N-hydroxy-4-(4-(9-isopropyl-2- morpholino-9H-purin-6-
yl)phenoxy)butanamide. LC-MS m/z 441 ([M + H].sup.+). .sup.1HNMR
(CD.sub.3OD) .delta. 8.72 (s, 1H), 8.39 (d, J = 8.4 Hz, 2H), 7.12
(d, J = 8.4 Hz, 2H), 4.96-4.85 (masked peak, 1H), 4.14 (t, J = 6.0
Hz, 2H), 3.96-3.82 (m, 8H), 2.36 (br s, 2H), 2.18-2.11 (m, 2H),
1.67 (d, J = 6.8 Hz, 6H). HRMS (ESI) m/z [M + H].sup.+ calcd for
C.sub.22H.sub.29N.sub.6O.sub.4, 441.2245; found, 441.2260. 14 12i
##STR00060## N-hydroxy-5-(4-(9-isopropyl-2- morpholino-9H-purin-6-
yl)phenoxy)pentanamide. LC-MS m/z 455 ([M + H].sup.+). .sup.1HNMR
(CD.sub.3OD) .delta. 8.50 (d, J = 8.4 Hz, 2H), 8.41 (s, 1H), 7.08
(d, J = 8.4 Hz, 2H), 4.94-4.84 (masked peak, 1H), 4.11 (t, J = 5.2
Hz, 2H), 3.94-3.81 (m, 8H), 2.21 (br s, 2H), 1.86 (br s, 4H), 1.65
(d, J = 6.8 Hz, 6H). 15 12j ##STR00061##
N-hydroxy-6-(4-(9-isopropyl-2- morpholino-9H-purin-6-
yl)phenoxy)hexanamide. LC-MS m/z 469 ([M + H].sup.+). .sup.1HNMR
(CD.sub.3OD) .delta. 8.65 (br s, 1H), 8.24 (d, J = 6.0 Hz, 2H),
6.98 (d, J = 7.2 Hz, 2H), 4.87 (masked peak, 1H), 3.98 (t, J = 6.2
Hz, 2H), 3.84-3.69 (m, 8H), 2.06 (br s, 2H), 1.78-1.73 (m, 2H),
1.63-1.59 (m, 2H), 1.55 (d, J = 6.8 Hz, 6H) 1.48-1.44 (m, 2H). 16
12k ##STR00062## N-hydroxy-7-(4-(9-isopropyl-2-
morpholino-9H-purin-6- yl)phenoxy)heptanamide. LC-MS m/z 483 ([M +
H].sup.+). .sup.1HNMR (CD.sub.3OD) .delta. 8.62 (br s, 1H), 8.25
(d, J = 6.4 Hz, 2H), 6.98 (d, J = 7.2 Hz, 2H), 4.78 (masked peak,
1H), 3.98 (t, J = 6.4 Hz, 2H), 3.84-3.70 (m, 8H), 2.02 (br s, 2H),
1.76-1.70 (m, 2H), 1.55 (d, J = 6.8 Hz, 8H), 1.47-1.42 (m, 2H),
1.40- 1.34 (m, 2H). 17 8b ##STR00063##
5-(6-(2-aminopyrimidin-5-yl)-2- morpholino-9H-purin-6-yl)-N-
hydroxypentanamide. LC-MS m/z 414.1 ([M + H].sup.+). HPLC purity
(254 nm): 98.6%. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.34 (s, 1H),
9.54 (s, 2H), 8.25 (s, 1H), 7.25 (s, 2H), 4.14 (t, J = 6.4 Hz, 2H),
3.79 (m, 4H), 3.72 (m, 4H), 1.99 (t, J = 7.2 Hz, 2H), 1.82 (m, 2H),
1.47 (m, 2H). 18 8c ##STR00064## 4-(6-(2-aminopyrimidin-5-yl)-2-
morpholino-9H-purin-9-yl)-N- hydroxybutanamide. LC-MS m/z 400.1 ([M
+ H].sup.+). HPLC purity (254 nm): 99.9%. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.34 (s, 1H), 9.55 (s, 2H), 8.25 (s, 1H),
7.50 (s, 2H), 4.14 (t, J = 6.4 Hz, 2H), 3.79 (m, 4H), 3.72 (m, 4H),
2.10 (m, 2H), 1.95 (m, 2H). 19 8d ##STR00065##
6-(6-(2-aminopyrimidin-5-yl)-2- morpholino-9H-purin-9-yl)-N-
hydroxyhexanamide. LC-MS m/z 428.1 ([M + H].sup.+). HPLC purity
(254 nm): 95.8%. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.32 (s, 1H),
9.54 (s, 2H), 8.24 (s, 1H), 7.42 (s, 2H), 4.12 (t, J = 6.8 Hz, 2H),
3.79 (m, 4H), 3.72 (m, 4H), 1.95 (t, J = 7.6 Hz, 2H), 1.82 (m, 2H),
1.54 (m, 2H), 1.23 (m, 2H). 20 12l ##STR00066##
N-hydroxy-5-((4-(9-isopropyl-2- morpholino-9H-purin-6-
yl)benzyl)oxy)pentanamide. LC-MS m/z 469 ([M + H].sup.+).
.sup.1HNMR (DMSO- d.sub.6) .delta. 10.37 (s, 1H), 8.77 (d, J = 8.0
Hz, 2H), 8.38 (s, 1H), 7.50 (d, J = 8.0 Hz, 2H), 4.80-4.73 (m, 1H),
4.55 (s, 2H), 3.84-3.74 (m, 8H), 3.47 (t, J = 6.0 Hz, 2H), 1.99 (t,
J = 6.0 Hz, 2H), 1.62 (masked peak, 10H). 21 12m ##STR00067##
N-hydroxy-6-((4-(9-isopropyl-2- morpholino-9H-purin-6-
yl)benzyl)oxy)hexanamide. LC-MS m/z 483 ([M + H].sup.+). .sup.1HNMR
(DMSO- d.sub.6) .delta. 10.37 (s, 1H), 8.76 (d, J = 8.0 Hz, 2H),
8.40 (s, 1H), 7.50 (d, J = 8.0 Hz, 2H), 4.78-4.73 (m, 1H), 4.55 (s,
2H), 3.84-3.74 (m, 8H), 3.47 (t, J = 6.0 Hz, 2H), 1.96 (t, J = 8.0
Hz, 2H), 1.57 (masked peak, 10H), 1.34-1.30 (m, 2H). HRMS (ESI) m/z
[M + H].sup.+ calcd for C.sub.25H.sub.35N.sub.6O.sub.4, 483.2714;
found, 483.2738. 22 12n ##STR00068##
N-hydroxy-7-((4-(9-isopropyl-2- morpholino-9H-purin-6-
yl)benzyl)oxy)heptanamide. LC-MS m/z 497 ([M + H].sup.+).
.sup.1HNMR (DMSO- d.sub.6) .delta. 10.37 (s, 1H), 8.76 (d, J = 8.0
Hz, 2H), 8.40 (s, 1H), 7.50 (d, J = 8.0 Hz, 2H), 4.80-4.73 (m, 1H),
4.55 (s, 2H), 3.84-3.74 (m, 8H), 3.47 (t, J = 6.0 Hz, 2H), 1.95 (t,
J = 6.0 Hz, 2H), 1.55 (masked peak, 10 H), 1.46-1.28 (m, 4H). 23
12o ##STR00069## N-hydroxy-5-((3-(9-isopropyl-2-
morpholino-9H-purin-6- yl)benzyl)oxy)pentanamide. LC-MS m/z 469 ([M
+ H].sup.+). .sup.1HNMR (DMSO- d.sub.6) .delta. 10.29 (s, 1H),
8.65-8.62 (masked peak, 2H), 8.31 (s, 1H), 7.49-7.40 (m, 2H),
4.72-4.66 (m, 1H), 4.50 (s, 2H), 3.77-3.66 (m, 8H), 3.41 (t, J =
6.0 Hz, 2H), 1.90 (t, J = 7.0 Hz, 2H), 1.50 (masked peak, 10 H).
HRMS (ESI) m/z [M + H].sup.+ calcd for
C.sub.24H.sub.33N.sub.6O.sub.4, 469.2558; found, 469.2570. 24 12p
##STR00070## N-hydroxy-2-(3-(9-isopropyl-2- morpholino-9H-purin-6-
yl)phenoxy)acetamide. LC-MS m/z 413.1 ([M + H].sup.+). HPLC purity
(254 nm): 97.9%. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.95 (s, 1H),
8.45 (d, J = 8.0 Hz, 1H), 8.42 (s, 1H), 8.32 (s, 1H), 7.49 (t, J =
8.0 Hz, 1H), 7.14 (dd, J = 8.4, 2.4 Hz, 1H), 4.76 (m, J = 6.8 Hz,
1H), 4.56 (s, 2H), 3.82 (m, 4H), 3.75 (m, 4H), 1.55 (d, J = 6.8 Hz,
6H). 25 12q ##STR00071## N-hydroxy-4-((3-(9-isopropyl-2-
morpholino-9H-purin-6- yl)phenoxy)methyl)benzamide. LC- MS m/z
489.2 ([M + H].sup.+). HPLC purity (254 nm): 91.2%. .sup.1H NMR
(DMSO-d.sub.6) .delta. 11.25 (s, 1H), 8.43 (d, J = 7.6 Hz, 1H),
8.39 (s, 1H), 8.35 (m, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.58 (d, J =
8.0 Hz, 1H), 7.49 (t, J = 8.0 Hz, 1H), 7.21 (dd, J = 7.8, 2.2 Hz,
1H), 5.28 (s, 2H), 4.76 (m J = 6.8 Hz, 1H), 3.77 (m, 4H), 3.74 (m,
4H), 1.55 (d, J = 6.8 Hz, 6H). HRMS (ESI) m/z [M + H].sup.+ calcd
for C.sub.26H.sub.28N.sub.6O.sub.4.sup.+, 489.2245; found,
489.2250. 26 12r ##STR00072## N-hydroxy-6-((3-(9-isopropyl-2-
morpholino-9H-purin-6- yl)benzyl)oxy)hexanamide. LC-MS m/z 483 ([M
+ H].sup.+). .sup.1HNMR (DMSO- d.sub.6) .delta. 10.34 (s, 1H),
8.72-8.70 (masked peak, 2H), 8.39 (s, 1H), 7.56-7.48 (m, 2H),
4.82-4.76 (m, 1H), 4.58 (s, 2H), 3.84-3.74 (m, 8H), 3.48 (t, J =
6.4 Hz, 2H), 1.95 (t, J = 7.2 Hz, 2H), 1.60-1.48 (masked peak, 10
H), 1.37-1.33 (m, 2H). 27 12s ##STR00073##
N-hydroxy-7-((3-(9-isopropyl-2- morpholino-9H-purin-6-
yl)benzyl)oxy)heptanamide. LC-MS m/z 497 ([M + H].sup.+).
.sup.1HNMR (DMSO- d.sub.6) .delta. 10.34 (br s, 1H), 8.73-8.69
(masked peak, 2H), 8.40 (s, 1H), 7.56- 7.48 (m, 2H), 4.80-4.74 (m,
1H), 4.57 (s, 2H), 3.84-3.74 (m, 8H), 3.48 (t, J = 6.8 Hz, 2H),
1.94 (t, J = 7.2 Hz, 2H), 1.56 (d, J = 6.8 Hz, 6H), 1.57-1.45
(masked peak, 4 H), 1.37-1.31 (m, 4H). 28 25a ##STR00074##
4-(6-(2-aminopyrimidin-5-yl)-8- (diethylamino)-2-morpholino-9H-
purin-9-yl)-N-hydroxybutanamide. LC-MS m/z 471.1 ([M + H].sup.+).
HPLC purity (254 nm): 95.5%. .sup.1H NMR (DMSO-d.sub.6) .delta.
10.40 (s, 1H), 9.47 (s, 2H), 7.56 (s, 2H), 4.01 (m, 1H), 3.72 (m,
8H), 3.38 (m, 4H), 1.99 (m, 4H), 1.17 (m, 6H). 29 12t ##STR00075##
N-hydroxy-4-(3-(9-isopropyl-2- (pyrrolidin-1-yl)-9H-purin-6-
yl)phenoxy)butanamide. LC-MS m/z 425.2 ([M + H].sup.+). HPLC purity
(254 nm): 98.8%. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.45 (s, 1H),
8.33-8.36 (m, 3H), 7.46 (t, J = 8.0 Hz, 1H), 7.11 (m, 1H), 4.73 (m,
J = 6.8 Hz, 1H), 4.06 (t, J = 6.4 Hz, 2H), 2.19 (t, J = 6.8 Hz,
6H), 1.98 (m, 6H), 1.56 (d, J = 6.8 Hz, 6H). HRMS (ESI) m/z [M +
H].sup.+ calcd for C.sub.22H.sub.28N.sub.6O.sub.3, 425.2296; found,
425.2305. 30 12u ##STR00076## 4-(3-(2-(diethylamino)-9-isopropyl-
9H-purin-6-yl) phenoxy)-N-hydroxybutanamide. LC- MS m/z 427.1 ([M +
H].sup.+). HPLC purity (254 nm): 98.2%. .sup.1H NMR (DMSO-d.sub.6)
.delta. 10.19 (s, 1H), 8.32 (s, 1H), 8.23-8.25 (m, 2H), 7.46 (t, J
= 8.0 Hz, 1H), 7.11 (dd-like, J = 8.0 Hz, 1H), 4.71 (m, J = 6.8 Hz,
1H), 4.04 (m, 2H), 3.89 (m, 4H), 2.17 (t, J = 6.8 Hz, 2H), 1.98 (m,
2H), 1.56 (d, J = 6.8 Hz, 6H), 1.20 (t, J = 8.0, 6H). 31 12v
##STR00077## 4-(3-(2-(dimethylamino)-9-isopropyl-
9H-purin-6-yl)phenoxy)-N- hydroxybutanamide. LC-MS m/z 399.1 ([M +
H].sup.+). HPLC purity (254 nm): 97.8%. .sup.1H NMR (DMSO-d.sub.6)
.delta. 8.37- 8.40 (m, 3H), 7.52 (t, J = 8.0 Hz, 1H), 7.16
(dt-like, J = 8.8 Hz, 1H), 4.79 (m, J = 6.8 Hz, 1H), 4.10 (m, 2H),
3.30 (s, 6H), 2.24 (m, 2H), 2.05 (m, 2H), 1.60 (d, J = 6.8 Hz, 6H).
HRMS (ESI) m/z [M + H].sup.+ calcd for
C.sub.20H.sub.26N.sub.6O.sub.3, 399.2140; found, 399.2137. 32 12w
##STR00078## N-hydroxy-2-((4-(9-isopropyl-2- morpholino-9H-purin-6-
yl)benzyl)oxy)acetamide. LC-MS m/z 427 ([M + H].sup.+). .sup.1HNMR
(DMSO-d.sub.6) .delta. 10.72 (br s, 1H), 8.77 (d, J = 8.4 Hz, 2H),
8.40 (s, 1H), 7.55 (d, J = 8.4 Hz, 2H), 4.80-4.73 (m, 1H), 4.63 (s,
2H), 3.94 (s, 2H), 3.84-3.74 (m, 8H), 1.55 (d, J = 6.8 Hz, 6H). 33
12x ##STR00079## N-hydroxy-2-(4-(9-isopropyl-2-
morpholino-9H-purin-6- yl)phenoxy)acetamide. LC-MS m/z 413 ([M +
H].sup.+). .sup.1HNMR (DMSO-d.sub.6) .delta. 10.92 (br s, 1H), 8.78
(d, J = 8.8 Hz, 2H), 8.35 (s, 1H), 7.13 (d, J = 8.8 Hz, 2H),
4.80-4.72 (m, 1H), 4.58 (s, 2H), 3.83-3.73 (m, 8H), 1.55 (d, J =
6.8 Hz, 6H). 34 12y ##STR00080## N-hydroxy-1-(3-(9-isopropyl-2-
morpholino-9H-purin-6- yl)benzyl)piperidine-4-carboxamide. LC-MS
m/z 480 ([M + H].sup.+). .sup.1HNMR (DMSO-d.sub.6) .delta. 10.63
(br s, 1H), 9.79 (br s, 1H), 8.91-8.88 (m, 1H), 8.80 (s, 1H), 8.41
(s, 1H), 7.68 (d, J = 4.8 Hz, 2H), 4.82-4.75 (m, 1H), 4.45 (br s,
2H), 3.85-3.76 (m, 8H), 3.46-3.43 (m, 2H), 3.04 (br s, 2H),
2.29-2.26 (m, 1H), 1.92-1.85 (m, 4H), 1.56 (d, J = 6.4 Hz, 6H).
HRMS (ESI) m/z [M + H].sup.+ calcd for
C.sub.25H.sub.34N.sub.7O.sub.3, 480.2718; found, 480.2716 35 12z
##STR00081## N-hydroxy-4-(((3-(9-isopropyl-2-
morpholino-9H-purin-6- yl)benzyl)amino)methyl)benzamide. LC-MS m/z
520 ([M + H].sup.+). .sup.1HNMR (DMSO-d.sub.6) .delta. 11.32 (br s,
1H), 8.91 (d, J = 8.8 Hz, 1H), 8.81 (s, 1H), 8.41 (s, 1H), 7.84 (d,
J = 8.4 Hz, 2H), 7.68-7.60 (m, 4H), 4.81-4.74 (m, 1H), 4.35-4.31
(m, 4H), 3.85-3.76 (m, 8H), 1.56 (d, J = 6.4 Hz, 6H). HRMS (ESI)
m/z [M + H].sup.+ calcd for C.sub.27H.sub.32N.sub.7O.sub.3,
502.2561; found, 502.2570. 36 12aa ##STR00082##
N-hydroxy-6-((3-(9-isopropyl-2- morpholino-9H-purin-6-
yl)benzyl)amino)hexanamide. LC-MS m/z 482 ([M + H].sup.+).
.sup.1HNMR (DMSO-d.sub.6) .delta. 10.42 (br s, 1H), 9.02 (br s,
2H), 8.90 (dt, J = 7.2, 1.6 Hz, 1H), 8.79 (s, 1H), 8.42 (s, 1H),
7.70-7.63 (m, 2H), 4.82-4.75 (m, 1H), 4.30 (t, J = 5.2 Hz, 2H),
3.87-3.75 (m, 8H), 3.00 (m, 2H), 1.98 (t, J = 7.2 Hz, 2H), 1.67-
1.61 (m, 2H), 1.57 (d, J = 6.4 Hz, 6H), 1.56-1.50 (overlapping,
2H), 1.34- 1.30 (m, 2H). HRMS (ESI) m/z [M + H].sup.+ calcd for
C.sub.25H.sub.36N.sub.7O.sub.3, 482.2874; found, 482.2891. 37 19a
##STR00083## N-hydroxy-7-(2-(3-hydroxyphenyl)-6-
morpholino-9H-purin-9- yl)heptanamide. LC-MS m/z 441.1 ([M +
H].sup.+). HPLC purity (254 nm): 87.0%. .sup.1H NMR (DMSO-d.sub.6)
.delta. 10.34 (s, 1H), 8.20 (s, 2H), 7.80-7.82 (m, 2H), 7.23 (t, J
= 8.0 Hz, 1H), 6.81 (dm, J = 8.0 Hz, 1H), 4.26 (m, 4H), 4.19 (m,
2H), 3.75 (m, 4H), 1.90 (t, J = 7.6 Hz, 2H), 1.83 (m, 2H), 1.45 (m,
2H), 1.25 (m, 4H). 38 9a ##STR00084##
6-((6-(2-aminopyrimidin-5-yl)-9-
isopropyl-9H-purin-2-yl)amino)-N- hydroxybutanamide. LC-MS m/z
400.1 ([M + H].sup.+). HPLC purity (254 nm): 91.6%. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.35 (s, 1H), 9.47 (s, 2H), 8.24 (s, 1H),
7.33 (s, 2H), 4.69 (m, J = 8.0 Hz, 1H), 3.35 (m, 2H), 2.52 (m, 2H),
1.96 (m, 2H), 1.59 (m, 2H), 1.53 (d, J = 6.8 Hz, 6H), 1.34 (m, 2H).
39 9b ##STR00085## 1-(6-(2-aminopyrimidin-5-yl)-9-
isopropyl-9H-purin-2-yl)-N hydroxypiperidine-4-carboxamide. LC-MS
m/z 442.1 ([M + H].sup.+). HPLC purity (254 nm): 93.1%. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.52 (s, 1H), 9.52 (s, 2H), 8.32 (s, 1H),
7.43 (s, 2H), 4.73 (m, J = 6.8 Hz, 1H), 2.93 (m, 2H), 2.32 (m, 1H),
1.71 (m, 2H), 1.60 (m, 2H), 1.54 (d, J = 6.8 Hz, 6H). 40 19b
##STR00086## N-hydroxy-7-(2-(4- hydroxymethyl)phenyl)-6-morpholino-
9H-purin-9-yl)heptanamide. LC-MS m/z 455.2 ([M + H].sup.+). HPLC
purity (254 nm): 97.0%. .sup.1H NMR (DMSO- d.sub.6) .delta. 10.34
(s, 1H), 8.36 (d, J = 8.4 Hz, 2H), 8.23 (s, 1H), 7.42 (d, J = 8.4
Hz, 2H), 4.57 (s, 2H), 4.30 (m, 2H), 4.23 (m, 4H), 3.78 (m, 4H),
1.94 (m, 2H), 1.86 (m, 2H), 1.48 (m, 2H), 1.29 (m, 4H). 41 19c
##STR00087## 7-(2-(2-aminopyrimidin-5-yl)-6-
morpholino-9H-purin-9-yl)-N- hydroxyheptanamide. LC-MS m/z 442.1
([M + H].sup.+). HPLC purity (254 nm): 97.8%. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.25 (s, 1H), 9.07 (s, 2H), 8.12 (s, 1H),
7.16 (bs, 2H), 4.25 (m, 4H), 4.13 (m, 2H), 3.68 (m, 4H), 1.85 (m,
2H), 1.77 (m, 2H), 1.40 (m, 2H), 1.20 (m, 4H). HRMS (ESI) m/z [M +
H].sup.+ calcd for C.sub.20H.sub.27N.sub.9O.sub.3, 442.2310; found,
442.2326. 42 12ab ##STR00088## N-hydroxy-2-((3-(9-isopropyl-2-
morpholino-9H-purin-6- yl)benzyl)oxy)acetamide. LC-MS m/z 427 ([M +
H].sup.+). .sup.1HNMR (CD.sub.3OD) .delta. 8.72-8.70 (m, 2H), 8.41
(s, 1H), 7.57- 7.54 (m, 2H), 4.80-4.73 (m, 1H), 4.65 (s, 2H), 3.95
(s, 2H), 3.83-3.74 (m, 8H), 1.56 (d, J = 6.8 Hz, 6H). HRMS (ESI)
m/z [M + H].sup.+ calcd for C.sub.21H.sub.27N.sub.6O.sub.4,
427.2088; found, 427.2101. 43 12ac ##STR00089##
N-hydroxy-4-(3-(2-morpholino-9- propyl-9H-purin-6-
yl)phenoxy)butanamide. LC-MS m/z 441.1 ([M + H].sup.+). HPLC purity
(254 nm): 99.6%. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.46 (s, 1H),
8.39 (s, 1H), 8.36 (d, J = 7.6 Hz, 1H), 8.30 (s, 1H), 7.46 (t, J =
8.0 Hz, 1H), ), 7.11 (d, J = 8.0 Hz, 1H), 4.11 (t, J = 7.2 Hz, 2H),
4.05 (t, J = 7.2 Hz, 2H), 3.82 (m, 4H), 3.75 (m, 4H), 2.19 (m, 2H),
2.00 (m, 2H), 1.87 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H). 44 12ad
##STR00090## N-hydroxy-4-(3-(9-propyl-2-
(pyrrolidin-1-yl)-9H-purin-6- yl)phenoxy)butanamide. LC-MS m/z
425.2 ([M + H].sup.+). HPLC purity (254 nm): 94.3% .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.47 (s, 1H), 8.37 (s, 1H), 8.34 (d, J =
7.6 Hz, 1H), 8.26 (s, 1H), 7.46 (t, J = 8.0 Hz, 1H), ), 7.11 (d, J
= 8.0 Hz, 1H), 4.11 (m, 2H), 4.06 (m, 2H), 3.62 (m, 4H), 2.19 (m,
2H), 1.98 (m, 6H), 1.88 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H). HRMS
(ESI) m/z [M + H].sup.+ calcd for C.sub.22H.sub.28N.sub.6O.sub.3,
425.2296; found, 425.2304. 45 12ae ##STR00091##
4-(3-(9-ethyl-2-morpholino-9H-purin-
6-yl)phenoxy)-N-hydroxybutnamide. LC-MS m/z 427.1 ([M + H].sup.+).
LC purity (254 nm): 90.3%. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.52
(s, 1H), 8.40 (s, 1H), 8.37-8.39 (m, 2H), 7.51 (t, J = 8.0 Hz, 1H),
), 7.16 (d, J = 8.0 Hz, 1H), 4.22 (t, J = 7.2 Hz, 2H), 4.10 (t, J =
7.2 Hz, 2H), 3.86 (m, 4H), 3.79 (m, 4H), 2.24 (m, 2H), 2.06 (m,
2H), 1.48 (t, J = 7.2 Hz, 3H). HP 46 12af ##STR00092##
4-(3-(9-ethyl-2-(pyrrolidin-1-yl)-9H- purin-6-yl)phenoxy)-N-
hydroxybutanamide. LC-MS m/z 411.2 ([M + H].sup.+). HPLC purity
(254 nm): 97.7%. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.46 (s, 1H),
8.36 (s, 1H), 8.35 (d, J = 7.6 Hz, 1H), 8.28 (s, 1H), 7.46 (t, J =
8.0 Hz, 1H), ), 7.11 (d, J = 8.0 Hz, 1H), 4.17 (m, 2H), 4.06 (m,
2H), 3.64 (m, 4H), 2.19 (m, 2H), 1.99 (m, 6H), 1.44 (t, J = 7.2 Hz,
3H). HRMS (ESI) m/z [M + H].sup.+ calcd for
C.sub.21H.sub.26N.sub.6O.sub.3, 411.2140; found, 411.2149. 47 19d
##STR00093## N-hydroxy-7-(2-(4- (methylsulfonamido)phenyl)-6-
morpholino-9H-purin-9- yl)heptanamide. LC-MS m/z 518.1 ([M +
H].sup.+). HPLC purity (254 nm): 96.7%. .sup.1H NMR (DMSO-d.sub.6)
.delta. 10.33 (s, 1H), 9.96 (s, 1H), 8.33 (d, J = 8.8 Hz, 2H), 8.21
(s, 1H), 7.29 (d, J = 8.8 Hz, 2H), 4.29 (m, 4H), 4.21 (m, 2H), 3.76
(m, 4H), 3.05 (s, 3H), 1.92 (m, 2H), 1.85 (m, 2H), 1.47 (m, 2H),
1.27 (m, 4H). 48 19e ##STR00094## 7-(2-(3-acetamidophenyl)-6-
morpholino-9H-purin-9-yl)-N- hydroxyheptanamide. LC-MS m/z 482.2
([M + H].sup.+). HPLC purity (254 nm): 98.8%. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.35 (s, 1H), 10.09 (s, 1H), 8.48 (s, 1H),
8.25 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H),
7.39 (t, J = 8.0 Hz, 1H), 4.32 (m, 4H), 4.23 (m, 2H), 3.79 (m, 4H),
2.09 (s, 3H), 1.94 (m, 2H), 1.87 (m, 2H), 1.48 (m, 2H), 1.29 (m,
4H). 49 19g ##STR00095## 3-(9-(7-(hydroxyamino)-7-oxoheptyl)-
6-morpholino-9H-purin-2- yl)benzamide. LC-MS m/z 468.2 ([M +
H].sup.+). HPLC purity (254 nm): 99.2%. .sup.1H NMR (DMSO-d.sub.6)
.delta. 10.33 (s, 1H), 8.84 (s, 1H), 8.53 (d, J = 8.0 Hz, 1H), 8.26
(s, 1H), 8.13 (s, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.56 (t, J = 8.0
Hz, 1H), 7.47 (s, 1H), 4.33 (m, 4H), 4.26 (m, 2H), 3.79 (m, 4H),
1.94 (m, 2H), 1.88 (m, 2H), 1.48 (m, 2H), 1.29 (m, 4H). 50 19h
##STR00096## 4-(9-(7-(hydroxyamino)-7-oxoheptyl)-
6-morpholino-9H-purin-2- yl)benzamide. LC-MS m/z 468.2 ([M +
H].sup.+). HPLC purity (254 nm): 99.3%. .sup.1H NMR (DMSO-d.sub.6)
.delta. 10.34 (s, 1H), 8.45 (d, J = 8.4 Hz, 2H), 8.27 (s, 1H), 8.05
(s, 1H), 7.97 (d, J = 8.4 Hz, 2H), 7.46 (s, 1H), 4.32 (m, 4H), 4.26
(m, 2H), 3.79 (m, 4H), 1.94 (m, 2H), 1.88 (m, 2H), 1.48 (m, 2H),
1.29 (m, 4H). 51 19i ##STR00097## N-hydroxy-7-(6-morpholino-2-(1H-
pyrazol-4-yl)-9H-purin-9- yl)heptanamide. LC-MS m/z 415.2 ([M +
H].sup.+). HPLC purity (254 nm): 97.3%. .sup.1H NMR (DMSO-d.sub.6)
.delta. 10.33 (s, 1H), 8.18 (s, 2H), 8.14 (s, 1H), 4.26 (m, 4H),
4.18 (m, 2H), 3.75 (m, 4H), 1.93 (m, 2H), 1.83 (m, 2H), 1.48 (m,
2H), 1.27 (m, 4H). HRMS (ESI) m/z [M + H].sup.+ calcd for
C.sub.19H.sub.26N.sub.8O.sub.3, 415.2201; found, 415.2214. 52 19j
##STR00098## N-hydroxy-7-(2-(6-methoxypyridin-3-
yl)-6-morpholino-9H-purin-9- yl)heptanamide. LC-MS m/z 456.1 ([M +
H].sup.+). HPLC purity (254 nm): 98.8%. .sup.1H NMR (DMSO-d.sub.6)
.delta. 10.34 (s, 1H), 9.15 (s, 1H), 8.57 (d, J = 8.0 Hz, 1H), 8.22
(s, 1H), 6.92 (d, J = 8.4 Hz, 1H), 4.30 (m, 4H), 4.22 (m, 2H), 3.93
(s, 3H), 3.77 (m, 4H), 1.93 (m, 2H), 1.86 (m, 2H), 1.48 (m, 2H),
1.28 (m, 4H). HRMS (ESI) m/z [M + H].sup.+, calcd for
C.sub.22H.sub.29N.sub.7O.sub.4, 456.2354; found, 456.2369. 53 19k
##STR00099## 7-(2-(6-aminopyridin-3-yl)-6-
morpholino-9H-purin-9-yl)-N- hydroxyheptanamide. LC-MS m/z 441.2
([M + H].sup.+). HPLC purity (254 nm): 97.3%. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.36 (s, 1H), 8.76 (m, 2H), 8.42 (s, 2H),
8.26 (s, 1H), 7.11 (d, J = 9.6 Hz, 1H), 4.30 (m, 4H), 4.22 (m, 2H),
3.77 (m, 4H), 1.93 (m, 2H), 1.85 (m, 2H), 1.47 (m, 2H), 1.28 (m,
4H). HRMS (ESI) m/z [M + H].sup.+ calcd for
C.sub.21H.sub.29N.sub.8O.sub.3, 441.2357; found, 441.2374. 54 19l
##STR00100## 7-(2-(1H-indazol-6-yl)-6-morpholino- 9H-purin-9-yl)-N-
hydroxyheptanamide. LC-MS m/z 465.2 ([M + H].sup.+). HPLC purity
(254 nm): 98.0%. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.38 (s, 1H),
8.60 (s, 1H), 8.25 (s, 1H), 8.22 (d, J = 8.8 Hz, 1H), 8.12 (s, 1H),
7.83 (d, J = 8.8 Hz, 1H), 4.34 (m, 4H), 4.27 (m, 2H), 3.80 (m, 4H),
1.95 (m, 2H), 1.90 (m, 2H), 1.51 (m, 2H), 1.32 (m, 4H). 55 19m
##STR00101## N-hydroxy-7-(2-(2-methoxypyrimidin-
5-yl)-6-morpholino-9H-purin-9- yl)heptanamide. LC-MS m/z 457.2 ([M
+ H].sup.+). HPLC purity (254 nm): 97.5%. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.35 (s, 1H), 9.39 (s, 2H), 8.25 (s, 1H),
4.28 (m, 4H), 4.22 (m, 2H), 3.99 (s, 3H), 3.76 (m, 4H), 1.93 (m,
2H), 1.85 (m, 2H), 1.47 (m, 2H), 1.28 (m, 4H). HRMS (ESI) m/z [M +
H].sup.+, calcd for C.sub.21H.sub.29N.sub.8O.sub.4, 457.2306;
found, 457.2327. 56 19n ##STR00102## 6-(2-(6-aminopyridin-3-yl)-6-
morpholino-9H-purin-9-yl)-N- hydroxyhexanamide. LC-MS m/z 427.2 ([M
+ H].sup.+). HPLC purity (254 nm): 93.3%. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.27 (s, 1H), 8.68-8.71 (m, 2H), 8.37 (bs,
2H), 8.18 (s, 1H), 7.02 (d, J = 8.8 Hz, 1H), 4.22 (m, 4H), 4.14 (t,
J = 6.8 Hz, 2H), 3.69 (m, 4H), 1.87 (m, 2H), 1.78 (m, 2H), 1.49 (m,
2H), 1.16 (m, 2H). 57 19o ##STR00103##
N-hydroxy-6-(2-(6-methoxypyridin-3- yl)-6-morpholino-9H-purin-9-
yl)hexanamide. LC-MS m/z 442.2 ([M + H].sup.+). HPLC (DMSO-d.sub.6)
.delta. 10.33 (s, 1H), 9.15 (s, 1H), 8.60 (d, J = 8.4 Hz, 1H), 8.22
(s, 1H), 6.92 (d, J = 8.4 Hz, 1H), 4.25 (m, 4H), 4.22 (t, J = 6.8
Hz, 2H), 3.93 (s, 3H), 3.77 (m, 4H), 1.94 (t, J = 6.8 Hz, 2H), 1.87
(m, 2H), 1.57 (m, 2H), 1.25 (m, 2H). HRMS (ESI) m/z [M + H].sup.+
calcd for C.sub.21H.sub.28N.sub.7O.sub.4, 442.2197; found,
442.2204. 58 19p ##STR00104## N-hydroxy-5-(2-(6-methoxypyridin-3-
yl)-6-morpholino-9H-purin-9- yl)pentanamide. LC-MS m/z 428.1 ([M +
H].sup.+). HPLC purity (254 nm): 92.3%. .sup.1H NMR (DMSO-d.sub.6)
.delta. 10.38 (s, 1H), 9.16 (s, 1H), 8.58 (d, J = 8.8 Hz, 1H), 8.22
(s, 1H), 6.92 (d, J = 8.8 Hz, 1H), 4.30 (m, 4H), 4.22 (t, J = 6.8
Hz, 2H), 3.93 (s, 3H), 3.77 (m, 4H), 2.01 (t, J = 7.2 Hz, 2H), 1.84
(m, 2H), 1.49 (m, 2H). HRMS (ESI) m/z [M + H].sup.+, calcd for
C.sub.20H.sub.26N.sub.7O.sub.4, 428.2041; found, 428.2042. 59 19q
##STR00105## 5-(2-(6-aminopyridin-3-yl)-6-
morpholino-9H-purin-9-yl)-N- hydroxypentanamide. LC-MS m/z 413.2
([M + H].sup.+). HPLC (254 nm): 80.5%. 60 19r ##STR00106##
4-((2-(6-aminopyridin-3-yl)-6- morpholino-9H-purin-9-yl)methyl)-N-
hydroxybenzamide. LC-MS m/z 447.1 ([M + H].sup.+). HPLC purity (254
nm): 92.1%. .sup.1H NMR (DMSO-d.sub.6) .delta. 11.17 (s, 1H), 9.01
(s, 1H), 8.77 (s, 1H), 8.67 (d, J = 8.4 Hz, 1H), 8.35 (s, 1H), 7.98
(bs, 1H), 7.69 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 6.97
(d, J = 8.4 Hz, 1H), 5.49 (s, 2H), 4.28 (m, 4H), 3.74 (m, 4H). 61
17b ##STR00107## (E)-3-(4-(((2-(2-(2-aminopyrimidin-5-
yl)-6-morpholino-9H-purin-9- yl)ethyl)amino)methyl)phenyl)-N-
hydroxyacrylamide. LC-MS m/z 531 ([M + H].sup.+). .sup.1HNMR
(DMSO-d.sub.6) .delta. 10.81 (br s, 1H), 9.16 (s, 2H), 9.02 (br s,
2H), 8.17 (s, 1H), 7.58 (d, J = 8.0 Hz, 2H), 7.48-7.43 (m, 3H),
7.16 (br s, 2H), 6.49 (d, J = 16.0 Hz, 1H), 4.58 (t, J = 5.2 Hz,
2H), 4.28 (t-like, 4H), 3.77-3.58 (m, 8H). HRMS (ESI) m/z [M +
H].sup.+, calcd for C.sub.29H.sub.29N.sub.10O.sub.3, 517.2419;
found, 517.2421. 62 12ag ##STR00108## 4-(2-fluoro-5-(9-isopropyl-2-
morpholino-9H-purin-6-yl)phenoxy)- N-hydroxybutanamide. LC-MS m/z
459.2 ([M + H].sup.+). HPLC purity (254 nm): 94.7%. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.48 (s, 1H), 8.62 (d, J = 7.2 Hz, 1H),
8.41 (m, 2H), 7.40 (t, J = 8.4 Hz, 1H), 4.76 (m, J = 6.8 Hz, 1H),
4.16 (m, 2H), 3.81 (m, 4H), 3.75 (m, 4H), 2.20 (m, 2H), 2.04 (m,
2H), 1.55 (d, J = 6.8 Hz, 6H). HRMS (ESI) m/z [M + H].sup.+, calcd
for C.sub.22H.sub.28FN.sub.6O.sub.4, 459.2151; found, 459.2154. 63
12ah ##STR00109## 4-(2-fluoro-5-(9-isopropyl-2-
(pyrrolidin-1-yl)-9H-purin-6- yl)phenoxy)-N-hydroxybutanamide.
LC-MS m/z 443.1 ([M + H].sup.+). HPLC purity (254 nm): 98.6%.
.sup.1H NMR (DMSO-d.sub.6) .delta. 10.48 (s, 1H), 8.61 (d, J = 8.4
Hz, 1H), 8.42 (m, 1H), 8.34 (s, 1H), 7.41 (t, J = 8.4 Hz, 1H), 4.74
(m, J = 6.8 Hz, 1H), 4.16 (m, 2H), 3.63 (m, 4H), 2.21 (m, 2H), 2.01
(m, 2H), 1.98 (m, 4H), 1.56 (d, J = 6.8 Hz, 6H). HRMS (ESI) m/z [M
+ H].sup.+, calcd for C.sub.22H.sub.28FN.sub.6O.sub.3, 443.2201;
found, 443.2216. 64 19s ##STR00110##
6-(2-(4-fluoro-3-hydroxyphenyl)-6- morpholino-9H-purin-9-yl)-N-
hydroxyhexanamide. LC-MS m/z 445.2 ([M + H].sup.+). HPLC purity
(254 nm): 98.0%. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.36 (s, 1H),
8.22 (s, 1H), 8.04 (d, J = 7.2 Hz, 1H), 7.85 (m, 1H), 7.21 (t, J =
8.4 Hz, 2H), 4.25 (m, 4H), 4.22 (m, 2H), 3.78 (m, 4H), 1.95 (m,
2H), 1.87 (m, 2H), 1.57 (m, 2H), 1.24 (m, 2H). 65 19t ##STR00111##
6-(2-(4-fluoro-3-(2- hydroxyethoxy)phenyl)-6-morpholino-
9H-purin-9-yl)-N-hydroxyhexanamide. LC-MS m/z 489.2 ([M +
H].sup.+). HPLC (254 nm): 90.3%. 66 12ai ##STR00112##
4-(2-fluoro-5-(9-propyl-2-(pyrrolidin-
1-yl)-9H-purin-6-yl)phenoxy)-N- hydroxybutanamide. LC-MS m/z 443.2
([M + H].sup.+). HPLC purity (254 nm): 99.3%. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.46 (s, 1H), 8.65 (d, J = 8.4 Hz, 1H),
8.45 (m, 1H), 8.22 (s, 1H), 7.40 (t, J = 8.8 Hz, 1H), 4.16 (m, 2H),
4.10 (m, 2H), 2.20 (m, 2H), 2.03 (m, 2H), 1.98 (m, 4H), 1.87 (m,
2H), 0.87 (t, J = 7.6 Hz, 3H). 67 19u ##STR00113##
7-(2-(4-fluoro-3-(2- hydroxyethoxy)phenyl)-6-morpholino-
9H-purin-9-yl)-N- hydroxyheptanamide. LC-MS m/z 503.1 ([M +
H].sup.+). HPLC purity (254 nm): 97.3%. .sup.1H NMR (DMSO-d.sub.6)
.delta. 10.26 (s, 1H), 8.16 (s, 1H), 8.04 (d, J = 8.8 Hz, 1H), 7.92
(m, 1H), 7.23 (t, J = 8.8 Hz, 1H), 4.22 (m, 4H), 4.16 (m, 2H), 4.10
(m, 2H), 3.73 (m, 2H), 3.70 (m, 4H), 1.85 (m, 2H), 1.79 (m, 2H),
1.40 (m, 2H), 1.21 (m, 4H). HRMS (ESI) m/z [M + H]+ calcd for
C.sub.24H.sub.32FN.sub.6O.sub.5, 503.2413; found, 503.2432. 68 12aj
##STR00114## 4-(5-(9-ethyl-2-morpholino-9H-purin-
6-yl)-2-fluorophenoxy)-N- hydroxybutanamide. LC-MS m/z 445.1 ([M +
H].sup.+). HPLC purity (254 nm): 98.8%. .sup.1H NMR (DMSO-d.sub.6)
.delta. 10.46 (s, 1H), 8.59 (d, J = 8.4 Hz, 1H), 8.40 (m, 1H), 8.30
(s, 1H), 7.37 (t, J = 8.4 Hz, 1H), 4.15 (m, 4H), 3.79 (m, 4H), 3.72
(m, 4H), 2.17 (m, 2H), 2.01 (m, 2H), 1.41 (t, J = 7.6 Hz, 3H). 69
12ak ##STR00115## 4-(2-fluoro-5-(2-morpholino-9-propyl-
9H-purin-6-yl)phenoxy)-N- hydroxybutanamide. LC-MS m/z 459.1 ([M +
H].sup.+). HPLC purity (254 nm): 99.0%. .sup.1H NMR (DMSO-d.sub.6)
.delta. 10.47 (s, 1H), 8.62 (d, J = 8.4 Hz, 1H), 8.42 (m, 1H), 8.31
(s, 1H), 7.39 (t, J = 8.4 Hz, 1H), 4.16 (m, 4H), 4.11 (m, 2H), 3.81
(m, 4H), 3.74 (m, 4H), 2.18 (m, 2H), 2.03 (m, 2H), 1.85 (m, 2H),
0.87 (t, J = 7.6 Hz, 3H). 70 19v ##STR00116##
7-(2-(4-fluoro-3-hydroxyphenyl)-6- morpholino-9H-purin-9-yl)-N-
hydroxyheptanamide. LC-MS m/z 459.1 ([M + H].sup.+). HPLC purity
(254 nm): 99.5%. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.35 (s, 1H),
9.97 (s, 1H), 8.19 (s, 1H), 8.02 (d, J = 8.8 Hz, 1H), 7.82 (m, 1H),
7.18 (t, J = 8.8 Hz, 1H), 4.23 (m, 4H), 4.19 (m, 2H), 3.75 (m, 4H),
1.92 (m, 2H), 1.84 (m, 2H), 1.46 (m, 2H), 1.26 (m, 4H).
71 17c ##STR00117## N-hydroxy-4-((((2-(2-(6-methoxy-
pyridin-3-yl)-6-morpholino-9H-purin-9-
yl)ethyl)amino)methyl)benzamide. LC-MS m/z 505 ([M + H].sup.+).
.sup.1HNMR (DMSO-d.sub.6) .delta. 11.27 (br s, 1H), 9.16 (d, J =
2.0 Hz, 1H), 8.54 (dd, J = 2.4 Hz, 8.8 Hz, 1H), 8.18 (s, 1H), 7.73
(d, J = 8.0 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.4 Hz,
1H), 4.59 (t, J = 5.6 Hz, 2H), 4.33-4.28 (m, 6H), 3.91 (s, 3H),
3.74 (t, J = 4.8 Hz, 4H), 3.58 (t-like, 2H). 72 19w ##STR00118##
N-hydroxy-7-(2-(3-(hydroxymethyl)-4-
methoxyphenyl)-6-morpholino-9H- purin-9-yl)heptanamide. LC-MS m/z
485.2 ([M + H].sup.+). HPLC purity (254 nm): 97.9%. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.32 (s, 1H), 8.44 (s, 1H), 8.28 (d, J =
8.4 Hz, 1H), 8.19 (s, 1H), 7.03 (d, J = 8.8 Hz, 1H), 4.55 (m, 2H),
4.30 (m, 4H), 4.22 (m, 2H), 3.85 (s, 3H), 3.77 (m, 4H), 1.93 (m,
2H), 1.86 (m, 2H), 1.48 (m, 2H), 1.28 (m, 4H). HRMS (ESI) m/z [M +
H]+ calcd for C.sub.24H.sub.33N.sub.6O.sub.5, 485.2507; found,
485.2506 73 19x ##STR00119## (N-hydroxy-7-(2-(3-(1-
(hydroxyimino)ethyl)-4- methoxyphenyl)-6-morpholino-9H-
purin-9-yl)heptanamide. LC-MS m/z 498.2 ([M + H].sup.+). HPLC
purity (254 nm): 99.1%. .sup.1H NMR (DMSO-d.sub.6) .delta. 11.35
(s, 1H), 10.33 (s, 1H), 8.70 (s, 1H), 8.40 (d, J = 8.8 Hz, 1H),
8.35 (s, 1H), 8.20 (s, 1H), 7.17 (d, J = 8.8 Hz, 1H), 4.29 (m, 4H),
4.22 (m, 2H), 3.90 (s, 3H), 3.77 (m, 4H), 1.93 (m, 2H), 1.86 (m,
2H), 1.47 (m, 2H), 1.28 (m, 4H). 74 19y ##STR00120##
N-hydroxy-7-(2-(3-(2- hydroxyethoxy)phenyl)-6-morpholino-
9H-purin-9-yl)heptanamide. LC-MS m/z 485.2 ([M + H].sup.+). HPLC
purity (254 nm): 99.1%. .sup.1H NMR (DMSO- d.sub.6) .delta. 10.31
(s, 1H), 8.22 (s, 1H), 7.95 (m, 1H), 7.90 (m, 1H), 7.35 (t, J = 8.0
Hz, 1H), 7.01 (m, 1H), 4.27 (m, 4H), 4.22 (m, 2H), 4.04 (m, 2H),
3.75 (m, 6H), 1.91 (m, 2H), 1.85 (m, 2H), 1.45 (m, 2H), 1.26 (m,
4H). 75 12al ##STR00121## N-hydroxy-4-(3-(7-isopropyl-2-
(pyrrolidin-1-yl)-7H-pyrrolo[2,3-
d]pyrimidin-4-yl)phenoxy)butanamide. LC-MS m/z 424.2 ([M +
H].sup.+). HPLC purity (254 nm): 97.9%. .sup.1H NMR (DMSO-d.sub.6)
.delta. 10.46 (s, 1H), 7.62 (m, 1H), 7.57 (s, 1H), 7.46 (t, J = 8.0
Hz, 1H), 7.39 (m, 1H), 7.12 (dd, J = 8.0, 2.0 Hz, 1H), 6.61 (d, J =
3.6 Hz, 1H), 4.89 (m, J = 6.8 Hz, 1H), 4.06 (t, J = 6.4 Hz, 2H),
3.62 (m, 4H), 2.16 (t, J = 6.8 Hz, 2H), 1.97 (m, 6H), 1.45 (d, J =
6.8 Hz, 6H). HRMS (ESI) m/z [M + H].sup.+ calcd for
C.sub.24H.sub.32N.sub.6O.sub.11, 424.2343; found, 424.2348. 76 12am
##STR00122## N-hydroxy-4-(3-(7-isopropyl-2-
morpholino-7H-pyrrolo[2,3- d]pyrimidin-4-yl)phenoxy)butanamide.
LC-MS m/z 440.1 ([M + H].sup.+). HPLC purity (254 nm): 97.8%;
.sup.1H NMR (DMSO-d.sub.6) .delta. 10.46 (s, 1H), 7.66 (m, 1H),
7.58 (s, 1H), 7.41-7.48 (m, 2H), 7.10 (dd, J = 8.0, 2.0 Hz, 1H),
6.65 (d, J = 4.0 Hz, 1H), 4.92 (m, J = 6.8 Hz, 1H), 4.04 (t, J =
6.4 Hz, 2H), 3.76 (m, 4H), 3.73 (m, 4H), 2.18 (m, 2H), 1.45 (d, J =
6.8 Hz, 6H), 1.18 (m, 2H); HRMS (ESI) m/z [M + H].sup.+ calcd for
C.sub.24H.sub.32N.sub.6O.sub.12, 440.2292; found, 440.2314. 77 19z
##STR00123## N-hydroxy-7-(2-(3- (hydroxymethyl)phenyl)-4-
morpholino-7H-pyrrolo[2,3- d]pyrimidin-7-yl)heptanamide. LC-MS m/z
454 ([M + H].sup.+). .sup.1HNMR (DMSO- d.sub.6) .delta. 10.33 (br
s, 1H), 8.35 (s, 1H), 8.28 (d, J = 7.6 Hz, 1H), 7.42-7.34 (m, 3H),
6.70 (d, J = 3.6 Hz, 1H), 4.58 (s, 2H), 4.24 (t, J = 7.2 Hz, 2H),
3.97-3.94 (m, 4H), 3.79-3.77 (m, 4H), 1.91 (t, J = 7.2 Hz, 2H),
1.84-1.77 (m, 2H), 1.49-1.42 (m, 2H), 1.33-1.23 (m, 4H). HRMS (ESI)
m/z [M + H].sup.+ calcd for C.sub.24H.sub.32N.sub.5O.sub.4,
454.2449; found, 454.2448. 78 19aa ##STR00124##
7-(2-(2-aminopyrimidin-5-yl)-4- morpholino-7H-pyrrolo[2,3-
d]pyrimidin-7-yl)-N- hydroxyheptanamide. LC-MS m/z 441 ([M +
H].sup.+). .sup.1HNMR DMSO-d.sub.6) .delta. 10.30 (s, 1H), 9.11 (s,
2H), 8.65 (s, 1H), 7.27 (d, J = 3.6 Hz, 1H), 6.97 (s, 2H), 6.65 (d,
J = 3.6 Hz, 1H), 4.19 (t, J = 6.8 Hz, 2H), 3.92-3.90 (m, 4H),
3.77-3.74 (m, 4H), 1.91 (t, J = 7.2 Hz, 2H), 1.78 (quintet, J = 7.2
Hz, 2H), 1.45 (quintet, J = 7.2 Hz, 2H), 1.33- 1.22 (m, 4H). 79
19ab ##STR00125## N-hydroxy-7-(2-(3-hydroxyphenyl)-4-
morpholino-7H-pyrrolo[2,3- d]pyrimidin-7-yl)heptanamide. LC-MS m/z
440 ([M + H].sup.+). .sup.1HNMR (DMSO- d.sub.6) .delta. 10.33 (br
s, 1H), 7.85-7.83 (m, 2H), 7.33 (d, J = 3.6 Hz, 1H), 7.25 (t, J =
8.0 Hz, 1H), 6.83-6.80 (m, 1H), 6.68 (d, J = 3.6 Hz, 1H), 4.23 (t,
J = 6.8 Hz, 2H), 3.95-3.93 (m, 4H), 3.79-3.77 (m, 4H), 1.92 (t, J =
7.2 Hz, 2H), 1.83- 1.76 (m, 2H), 1.49-1.42 (m, 2H), 1.33- 1.26 (m,
4H). HRMS (ESI) m/z [M + H].sup.+ calcd for
C.sub.23H.sub.30N.sub.5O.sub.4, 440.2292; found, 440.2306. 80 24b
##STR00126## 4-(((6-(2-aminopyrimidin-5-yl)-9-
isopropyl-2-morpholino-9H-purin-8- yl)amino)methyl)-N-
hydroxybenzamide. LC-MS m/z 505.2 ([M + H].sup.+). HPLC purity (254
nm): 98.9%. .sup.1H NMR (DMSO-d.sub.6) .delta. 11.09 (s, 1H), 9.28
(s, 2H), 7.81 (s, 1H), 7.77 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.4
Hz, 2H), 7.22 (bs, 1H), 4.59 (m, 3H), 3.63 (m, 4H), 3.60 (m, 4H),
1.52 (d, J = 6.8 Hz, 6H). 81 21a ##STR00127##
N-hydroxy-7-(4-(9-isopropyl-6- morpholino-9H-purin-2-
yl)phenoxy)heptanamide. LC-MS m/z 483.1 ([M + H].sup.+). HPLC
purity (254 nm): 98.1%. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.37
(s, 1H), 8.32 (d, J = 9.2 Hz, 2H), 8.27 (s, 1H), 7.01 (d, J = 8.8
Hz, 2H), 4.87 (m, J = 6.8 Hz, 1H), 4.29 (m, 2H), 4.01 (m, J = 6.4
Hz, 2H), 3.76 (m, 4H), 1.97 (t, J = 7.6 Hz, 2H), 1.73 (m, 2H), 1.57
(d, J = 6.4 Hz, 6H), 1.53 (m, 2H), 1.42 (m, 2H), 1.32 (m, 2H). HRMS
(ESI) m/z [M + H].sup.+ calcd for C.sub.25H.sub.34N.sub.6O.sub.4,
483.2715; found, 483.2730. 82 21b ##STR00128##
N-hydroxy-4-(4-(9-isopropyl-6- morpholino-9H-purin-2-
yl)phenoxy)butanamide. LC-MS m/z 441.1 ([M + H].sup.+). HPLC purity
(254 nm): 99.1%. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.37 (s, 1H),
8.25 (d, J = 8.8 Hz, 2H), 8.20 (s, 1H), 6.94 (d, J = 9.2 Hz, 2H),
4.79 (m, J = 6.8 Hz, 1H), 4.21 (m, 2H), 3.95 (m, J = 6.4 Hz, 2H),
3.70 (m, 4H), 2.10 (t, J = 7.2 Hz, 2H), 1.90 (m, 2H), 1.50 (d, J =
6.8 Hz, 6H). 83 21c ##STR00129## N-hydroxy-4-(3-(9-isopropyl-6-
morpholino-9H-purin-2- yl)phenoxy)butanamide. LC-MS m/z 441.2 ([M +
H].sup.+). HPLC purity (254 nm): 99.1%. .sup.1H NMR (DMSO-d.sub.6)
.delta. 10.46 (s, 1H), 8.33 (s, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.91
(s, 1H), 7.38 (t, J = 8.0 Hz, 1H), 7.03 (m, 1H), 4.89 (m, J = 6.8
Hz, 1H), 4.31 (m, 4H), 4.04 (m, J = 6.4 Hz, 2H), 3.77 (m, 4H), 2.18
(t, J = 7.2 Hz, 2H), 1.99 (m, 2H), 1.59 (d, J = 6.8 Hz, 6H). 84 21d
##STR00130## N-hydroxy-7-(3-(9-isopropyl-6- morpholino-9H-purin-2-
yl)phenoxy)heptanamide. LC-MS m/z 483.2 ([M + H].sup.+). HPLC
purity (254 nm): 99.3%. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.37
(s, 1H), 8.32 (s, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.91 (s, 1H), 7.38
(t, J = 8.0 Hz, 1H), 7.03 (m, 1H), 4.89 (m, J = 6.8 Hz, 1H), 4.30
(m, 4H), 4.04 (m, J = 6.4 Hz, 2H), 3.77 (m, 4H), 1.97 (t, J = 7.2
Hz, 2H), 1.75 (m, 2H), 1.59 (d, J = 6.8 Hz, 6H), 1.57 (m, 2H), 1.45
(m, 2H), 1.33 (m, 2H). HRMS (ESI) m/z [M + H].sup.+ calcd for
C.sub.25H.sub.34N.sub.6O.sub.4, 483.2175; found, 483.2727. 85 21e
##STR00131## N.sup.1-hydroxy-N.sup.8-(3-(9-isopropyl-6-
morpholino-9H-purin-2- yl)phenyl)octanediamide. LC-MS m/z 510.2 ([M
+ H].sup.+). HPLC purity (254 nm): 97.5%. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.37 (s, 1H), 10.01 (s, 1H), 8.48 (s, 1H),
8.32 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H),
7.38 (t, J = 8.0 Hz, 1H), 4.89 (m, J = 6.8 Hz, 1H), 4.32 (m, 4H),
3.78 (m, 4H), 2.34 (t, J = 7.2 Hz, 2H), 1.96 (t, J = 7.2 Hz, 2H),
1.59 (m, 8H), 1.51 (m, 2H), 1.31 (m, 4H). 86 27a ##STR00132##
1-(2-(2-aminopyrimidin-5-yl)-9- isopropyl-6-morpholino-9H-purin-8-
yl)-N-hydroxypiperidine-4- carboxamide. LC-MS m/z 483.2 ([M +
H].sup.+). HPLC purity (254 nm): 80.2%. .sup.1H NMR (DMSO-d.sub.6)
.delta. 10.26 (s, 1H), 9.09 (s, 2H), 7.24-7.38 (m, 2H), 4.51 (m, J
= 6.8 Hz, 1H), 4.19 (m, 4H), 3.73 (m, 4H), 2.90 (m, 2H), 1.76 (m,
4H), 1.64 (m, 6H). 87 32a ##STR00133##
6-(((2-(2-aminopyrimidin-5-yl)-9- ethyl-6-morpholino-9H-purin-8-
yl)methyl)amino)-N- hydroxyhexanamide. LC-MS m/z 485 ([M +
H].sup.+). .sup.1HNMR (DMSO-d.sub.6) .delta. 10.4 (br s, 1H), 9.16
(s, 2H), 7.29 (br s, 2H) 4.60 (t-like, J = 5.2 Hz, 2H), 4.31- 4.26
(m, 6H), 3.78 (t-like, J = 4.6 Hz, 4H), 3.15-2.70 (m, 2H), 1.98 (t,
J = 7.4 Hz, 2H), 1.75-1.66 (m, 2H), 1.58-1.50 (m, 2H), 1.39-1.30
(m, 5H). HRMS (ESI) m/z [M + H].sup.+ calcd for
C.sub.22H.sub.33N.sub.10O.sub.3, 485.2732; found, 485.2734. 88 32b
##STR00134## 1-((2-(2-aminopyrimidin-5-yl)-9-ethyl-
6-morpholino-9H-purin-8-yl)methyl)-
N-hydroxypiperidine-4-carboxamide. LC-MS m/z 483 ([M + H].sup.+).
.sup.1HNMR (DMSO-d.sub.6) .delta. 10.64 (br s, 1H), 9.17 (s, 2H),
7.35 (br s, 2H), 4.70 (s, 2H), 4.35-4.31 (m, 6H), 3.78 (t-like, J =
4.6 Hz, 4H), 3.68 (d-like, 2H), 3.16 (br s, 2H), 1.95-1.87 (m, 4H),
1.38 (t, J = 7.2 Hz, 3H). 89 35a ##STR00135##
(E)-3-(4-((((2-(2-aminopyrimidin-5-
yl)-9-ethyl-6-morpholino-9H-purin-8-
yl)methyl)(methyl)amino)methyl) phenyl)-N-hydroxyacrylamide. LC-MS
m/z 545 ([M + H].sup.+). .sup.1HNMR (CD.sub.3OD) .delta. 9.25 (s,
2H), 7.66-7.53 (dd-like, J = 7.6 Hz, 4H), 7.56 (masked peak, 1H),
6.51 (d, J = 16 Hz, 1H), 4.78 (s, 2H), 4.58 (br s, 2H), 4.39-4.29
(m, 6H), 3.87 (t-like, 4H), 3.07 (s, 3H), 1.43 (t, J = 6.8 Hz, 3H).
HRMS (ESI) m/z [M + H].sup.+ calcd for
C.sub.27H.sub.33N.sub.10O.sub.3, 545.2732; found, 545.2742. 90 35b
##STR00136## (E)-4-(9-ethyl-8-(((4-(3-
(hydroxyamino)-3-oxoprop-1-en-1- yl)benzyl)(methyl)amino)methyl)-6-
morpholino-9H-purin-2-yl)benzamide. LC-MS m/z 531 ([M + H].sup.+).
.sup.1HNMR (DMSO-d.sub.6) .delta. 10.86 (br s, 1H), 8.45 (d, J =
8.4 Hz, 2H), 8.06 (br s, 1H), 7.98 (d, J = 8.8 Hz, 2H), 7.67 (d, J
= 7.6 Hz, 1H), 7.56-7.47 (m, 4H), 6.53 (d, J = 15.6 Hz, 1H),
4.35-4.33 (m, 6H), 4.00 (br s, 4H), 3.83 (t-like, J = 4.4 Hz, 4H),
2.83 (br s, 3H), 1.39 (t, J = 7.2 Hz, 3H). HRMS (ESI) m/z [M +
H].sup.+ calcd for C.sub.30H.sub.35N.sub.8O.sub.4, 571.2776; found,
531.2799. 91 35c ##STR00137## (E)-3-(9-ethyl-8-(((4-(3-
(hydroxyamino)-3-oxoprop-1-en-1- yl)benzyl)(methyl)amino)methyl)-6-
morpholino-9H-purin-2-yl)benzamide. LC-MS m/z 571 ([M + H].sup.+).
.sup.1HNMR (DMSO-d.sub.6) .delta. 10.84 (br s, 1H), 8.86 (t, J =
1.6 Hz, 1H), 8.55 (dt, J = 1.2 Hz, 8.0 Hz, 1H), 8.14 (br s, 1H),
7.97 (dt like, J = 7.6 Hz, 1H), 7.68 (d, J = 7.6 Hz, 1H), 7.60-7.48
(m, 5H), 6.53 (d, J = 16.0 Hz, 1H), 4.35-4.34 (m, 6H), 4.20 (br s,
4H), 3.83 (t, J = 4.4 Hz, 4H), 2.87 (br s, 3H), 1.40 (t, J = 7.2
Hz, 3H). 92 35d ##STR00138## (E)-3-(4-((((2-(3-acetamidophenyl)-9-
ethyl-6-morpholino-9H-purin-8- yl)methyl)(methyl)amino)methyl)
phenyl)-N-hydroxyacrylamide. LC-MS m/z 585 ([M + H].sup.+).
.sup.1HNMR (CD.sub.3OD) .delta. 8.62 (br s, 1H), 8.16 (d, J = 7.6
Hz, 1H), 7.75-7.65 (m, 3H), 7.57-7.50 (m, 4H), 7.38 (t, J = 7.6 Hz,
1H), 4.80 (br s, 2H), 4.61 (br s, 2H), 4.42-4.30 (m, 6H), 3.89
(t-like, 4H), 3.10 (s, 3H), 2.17 (s, 3H), 1.45 (t, J = 7.2 Hz, 3H).
93 35e ##STR00139## (E)-3-(4-((((9-ethyl-2-(6-
methoxypyridin-3-yl)-6-morpholino-
9H-purin-8-yl)methyl)(methyl)amino)
methyl)phenyl)-N-hydroxyacrylamide. LC-MS m/z 559 ([M + H].sup.+).
.sup.1HNMR (DMSO-d.sub.6) .delta. 9.18 (d, J = 2.0 Hz, 1H), 8.60
(dd, J = 2.4 Hz, 8.4 Hz, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.56 (d, J
= 8.0 Hz, 2H), 7.49 (d, J = 16.0 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H),
6.53 (d, J = 15.6 Hz, 1H), 4.33-4.29 (m, 6H), 3.94 (s, 3H), 3.81
(overlapping peaks, 8H), 2.85 (br s, 3H), 1.38 (t, J = 6.8 Hz, 3H).
HRMS (ESI) m/z [M + H].sup.+ calcd for
C.sub.29H.sub.35N.sub.8O.sub.4, 559.2776; found, 559.2785. 94 35f
##STR00140## (E)-3-(4-((((9-ethyl-2-(4-
(methylsulfonamido)phenyl)-6- morpholino-9H-purin-8-
yl)methyl)(methyl)amino)methyl) phenyl)-N-hydroxyacrylamide. LC-MS
m/z 621 ([M + H].sup.+). 95 ##STR00141##
(E)-3-(4-((((2-chloro-9-ethyl-6- morpholino-9H-purin-8-
yl)methyl)(methyl)amino)methyl) phenyl)-N-hydroxyacrylamide. LC-MS
m/z 486 ([M + H].sup.+). .sup.1HNMR (DMSO- d.sub.6) .delta. 7.65
(d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.48 (overlapping,
d, J = 15.6 Hz, 1H), 6.52 (d, J = 16.0 Hz, 1H), 4.21-4.16 (m, 10H),
3.77 (t-like, 4H), 2.80 (br s, 3H), 1.29 (t, J = 7.2 Hz, 3H). 96
35g ##STR00142## 4-((((9-ethyl-2-(6-methoxypyridin-3-
yl)-6-morpholino-9H-purin-8- yl)methyl)(methyl)amino)methyl)-N-
hydroxybenzamide. LC-MS m/z 533 ([M + H].sup.+). .sup.1HNMR
(DMSO-d.sub.6) .delta. 11.32 (br s, 1H), 9.15 (d, J = 2.0 Hz, 1H),
8.57 (dd, J = 2.4 Hz, 8.8 Hz, 1H), 7.83 (d, J = 8.0 Hz, 2H), 7.59
(d, J = 7.6 Hz, 2H), 6.91 (d, J = 8.8 Hz, 1H), 4.63 (br s, 2H),
4.32-4.26 (m, 8H), 3.91 (s, 3H), 3.78 (t, J = 4.4 Hz, 4H), 2.77 (br
s, 3H), 1.35 (t, J = 6.8 Hz, 3H). HRMS (ESI) m/z [M + H].sup.+
calcd for C.sub.27H.sub.33N.sub.8O.sub.4. 533.2619; found,
533.2640. 97 35i ##STR00143## 6-(((9-ethyl-2-(3-
(hydroxymethyl)phenyl)-6- morpholino-9H-purin-8-
yl)methyl)(methyl)amino)-N- hydroxyhexanamide. LC-MS m/z 512 ([M +
H].sup.+). .sup.1HNMR (DMSO-d.sub.6) .delta. 10.38 (br s, 1H), 8.36
(s, 1H), 8.29 (dt, J = 1.6 Hz, 7.2 Hz, 1H), 7.46-7.40 (m, 2H), 4.59
(s, 2H), 4.37-4.31 (m, 6H), 3.79 (t, J = 4.8 Hz, 4H), 3.36 (br s,
2H), 3.18 (br s, 2H), 2.96 (s, 3H), 1.82-1.74 (m, 2H), 1.57-1.50
(m, 2H), 1.40 (t, J = 7.2 Hz, 3H), 1.32-1.28 (m, 2H). HRMS (ESI)
m/z [M + H].sup.+ calcd for C.sub.26H.sub.38N.sub.7O.sub.4,
512.2980; found, 512.2983. 98 ##STR00144##
7-(2,6-dimorpholino-9H-purin-9-yl)-N- hydroxyheptanamide. LC-MS m/z
434.2 ([M + H].sup.+). HPLC purity (254 nm): 96.5%. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.33 (s, 1H), 7.89 (s, 1H), 4.12 (m, 4H),
4.02 (m, 2H), 3.70 (m, 4H), 3.64 (m, 4H), 1.92 (m, 2H), 1.76 (m,
2H), 1.46 (m, 2H), 1.23 (m, 4H). 99 ##STR00145##
7-(2,6-di(pyrrolidin-1-yl)-9H-purin-9- yl)-N-hydroxyheptanamide.
LC-MS m/z 402.2 ([M + H].sup.+). HPLC purity (254 nm): 97.1%.
.sup.1H NMR (DMSO-d.sub.6) .delta. 10.37 (s, 1H), 8.31 (s, 1H),
4.15 (m, 2H), 3.94 (m, 2H), 3.67 (m, 2H), 3.53 (m, 4H), 1.95 (m,
10H), 1.77 (m, 2H), 1.47 (m, 2H), 1.25 (m, 2H). 100 ##STR00146##
4-(3-(9-cyclopentyl-2-morpholino-9H- purin-6-yl)phenoxy)-N-
hydroxybutanamide. LC-MS m/z 467.1 ([M + H].sup.+). HPLC purity
(254 nm): 95.6%. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.39 (s, 1H),
8.29 (s, 1H), 8.26 ((m, 2H), 7.38 (t, 1H), 7.04
(dd, J = 8.0, 2.4 Hz, 1H), 4.78 (m, 1H), 3.98 (m, 2H), 3.74 (m,
4H), 3.67 (m, 4H), 2.10 (m, 4H), 1.94 (m, 4H), 1.82 (m, 2H), 1.64
(m, 2H). 101 ##STR00147## N-hydroxy-4-(3-(2-morpholino-9-
(pentan-3-yl)-9H-purin-6- yl)phenoxy)butanamide. LC-MS m/z 469.1
([M + H].sup.+). HPLC purity (254 nm): 97.8%. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.46 (s, 1H), 8.35-8.38 (m, 3H), 7.46 (t,
1H), 7.11 (dd, J = 8.0, 2.2 Hz, 1H), 4.29 (m, 1H), 4.06 (t, J = 6.4
Hz, 2H), 3.79 (m, 4H), 3.75 (m, 4H), 2.18 (m, 2H), 2.01 (m, 4H),
1.91 (m, 2H), 0.74 (t, J = 7.2 Hz, 6H). 102 ##STR00148##
7-(2-(2-aminopyrimidin-5-yl)-6-(4-
(hydroxymethyl)piperidin-1-yl)-9H-
purin-9-yl)-N-hydroxyheptanamide. LC-MS m/z 470.1 ([M + H].sup.+).
HPLC purity (254 nm): 97.2%. .sup.1H NMR (DMSO-d.sub.6) .delta.
9.16 (s, 2H), 8.16 (s, 1H), 4.80 (m, 2H), 4.18 (t, J = 7.2 Hz, 2H),
3.28 (m, 2H), 3.09 (m, 2H), 1.92 (m, 2H), 1.83 (m, 5H), 1.48 (m,
2H), 1.20 (m, 6H). 103 ##STR00149## N-hydroxy-7-(2-(3-
(hydroxymethyl)phenyl)-6-(4- (hydroxymethyl)piperidin-1-yl)-9H-
purin-9-yl)heptanamide. LC-MS m/z 483.1 ([M + H].sup.+). HPLC
purity (254 nm): 86.2%. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.33
(s, 1H), 8.34 (s, 1H), 8.26 (d, J = 7.2 Hz, 1H), 8.20 (s, 1H), 7.41
(m, 2H), 5.50 (m, 2H), 4.59 (s, 2H), 4.22 (t, J = 6.8 Hz, 2H), 3.29
(m, 2H), 3.11 (m, 2H), 1.93 (m, 2H), 1.85 (m, 5H), 1.48 (m, 2H),
1.20 (m, 6H). 104 ##STR00150## 7-(2-(2-aminopyrimidin-5-yl)-6-(4-
hydroxypiperidin-1-yl)-9H-purin-9-yl)- N-hydroxyheptanamide. LC-MS
m/z 456.1 ([M + H].sup.+). HPLC purity (254 nm): 99.9%. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.33 (bs, 1H), 9.13 (s, 2H), 8.15 (s, 1H),
7.26 (bs, 2H), 4.88 (m, 1H), 4.19 (t, J = 6.8 Hz, 2H), 3.81 (m,
2H), 3.67 (m, 2H), 1.93 (m, 2H), 1.84 (m, 4H), 1.48 (m, 4H), 1.28
(m, 4H). 105 ##STR00151## N-hydroxy-7-(2-(3-(hydroxymethyl)
phenyl)-6-(4-hydroxypiperidin- 1-yl)-9H-purin-9-yl)heptanamide.
LC-MS m/z 469.1 ([M + H].sup.+). HPLC purity (254 nm): 93.3%.
.sup.1H NMR (DMSO-d.sub.6) .delta. 10.34 (s, 1H), 8.35 (s, 1H),
8.27 (m, 1H), 8.21 (s, 1H), 7.42 (m, 2H), 4.95 (m, 1H), 4.60 (s,
2H), 4.23 (m, 2H), 1.93 (m, 2H), 1.91 (m, 4H), 1.48 (m, 4H), 1.30
(m, 4H).
Example 10: Enzyme Assays
HDAC Enzyme Assay
[0336] HeLa nuclear extracts are used as the source of HDACs in
routine HDAC inhibition assays. The recombinant HDAC enzymes, HDAC1
(Cat #5005), HDAC3/NcoR2 (Cat #50003), HDAC4 (Cat #50004), HDAC6
(Cat #50006), HDAC8 (Cat #50008) were purchased from BPS Bioscience
Inc., United States. HDAC4 (#H86-31G-10), HDAC5 (Cat #H87-31G),
HDAC9 (Cat #H91-31G), HDAC10 (Cat #H92-31G), and HDAC11 (Cat
#H93-30G) were purchased from SignalChem, Canada. The assay is
performed in 96-well format (black NBS half-area 96-well plate,
Corning #3993) using a fluorescent-based HDAC activity assay.
Substrates Boc-Lys(Ac)-AMC (Cat #1-1875) for HeLa nuclear extracts,
HDACs 1, 2, 3, 6, and 10, Boc-Lys(Tfa)-AMC (Cat #1-1985) for HDACs
4, 5, 7, 8, 9 and 11 were purchased from Bachem AG, Switzerland.
The reaction mixture (50 .mu.L/well) is composed of assay buffer,
containing 25 mM Tris, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM
MgCl.sub.2, 0.1 mg/mL BSA, test compounds, an appropriate
concentration of enzyme, and 50 .mu.M of substrate and incubates at
room temperature for 2 h. The reaction is stopped by addition of
developer [50 .mu.L/well, containing trypsin (Cat #T4799,
Sigma-Aldrich), 2 mg/mL, 50 mM tris pH 8.0, and LAQ824 (CAS
404951-53-7), 4.5 .mu.M] and incubated at 37.degree. C. for 30 min.
The fluorescence is detected at the excitation wavelength of 360 nm
and emission wavelength of 460 nm using a BioTek Synergy H4 Hybrid
Multi-Mode Microplate Reader and raw data are processed using
BioTek's Gen5 (v2.03.01) software. The IC.sub.50s are reported in
Table 1, vorinostat (SAHA) is used as positive control which was
made as our previously reported (Wang, et al. J. Med. Chem. 2011,
54, 4694-4720).
Kinase Enzyme Assay.
[0337] Lipid kinases PI3K.alpha.: Cat # PV4788 from Invitrogen, or
Cat #40620 from BPS Bioscience Inc., United States or Cat # P27-18H
from SignalChem, Canada; p110.alpha.(H1047R)/p85.alpha. (Cat
#40641) from BPS, p110.alpha.(E545K)/p85.alpha. (Cat #P27-15H) from
SignalChem; PI3K.beta.: Cat # P28-10H (SignalChem), Cat #40622
(BPS), PI3K6: Cat # P30-10H (SignalChem) were used in the
ADP-Glo.TM. Kinase Assay (Promega). PI3K inhibitors GDC-0941 and
wortmannin were purchased from LC Laboratories (165 New Boston
Street Woburn, Mass. 01801, United States) as powder and then
prepared in DMSO as 10 mM stock. Dual inhibitor of PI3k and mTOR
GDC-0980 was purchased from Selleck Chemicals (2626 S Loop W #225,
Houston, Tex. 77054, United States) as 10 mM stock in DMSO.
Serially diluted compounds solutions (5 .mu.L/well, 3-fold,
8-concentration) was added to a white NBS half-area 96-well plate
(Corning #3992). Lipid PIP2:PS (1:3) mixture (0.167 mg/mL: 0.5
mg/mL) in Lipid Dilution Buffer (25 mM HEPES, pH7.5, 0.5 mM EGTA)
was diluted (1:1) with Reaction Buffer (3.33.times.) (159 mM HEPES,
pH7.5, 87 mM NaCl, 9.5 mM MgCl.sub.2, 0.08 mg/mL BSA) to make a
1.67.times. working solution, PI3K enzyme was diluted with the
1.67.times. working solution and 15 .mu.L/well was used for
reaction. ATP (125 .mu.M, 5 .mu.L/well) in DI water was added to
initiate the reaction. After reaction at room temp for 1 h, the
reaction was stopped by addition of ADP-Glo solution (25
.mu.L/well) and incubated at room temp for 40 min, then kinase
detection solution (50 .mu.L/well) was added and incubated for 40
min, the luminescence was read on a Biotek Synergy H4 Hybrid
Multi-Mode Microplate Reader and raw data are processed using
BioTek's Gen5 (v2.03.01) software. The IC50s are reported in Table
1, GDC-0941, GDC-0980 and wortmannin are used as positive
controls.
[0338] IC.sub.50 is defined as the concentration of compound
required for 50% inhibition of enzyme activity. Definition of the
potency: "I": 1 .mu.M<IC.sub.50.ltoreq.10 .mu.M. "II": 0.1
.mu.M<IC.sub.50.ltoreq.1 .mu.M. "III": 0.01
.mu.M<IC.sub.50.ltoreq.0.1 .mu.M. "IV": IC.sub.50.ltoreq.0.01
.mu.M
TABLE-US-00006 TABLE 3 In Vitro Enzymatic IC.sub.50 (.mu.M) HDAC
PI3K.alpha. PI3K.alpha. EX (HeLa) HDAC1 HDAC6 PI3K.alpha. (E545K)
(H1047R) PI3K.beta. 1 III III III I I I 2 IV IV IV III III III II 3
III III III III 4 III 5 III 6 II III II III 7 II III III III III
III III 8 III 9 II 10 III II I 11 III 12 III 13 I 14 II 15 II 16 II
17 I I 18 I 19 III III 20 II 21 II 22 I 23 III III 24 I I 28 I I 29
II III 30 II I 31 III III 32 I 33 I 34 I 35 II 36 III 37 II 38 IV
IV IV III III III II 39 III 40 I 41 IV IV IV I 42 II 43 III II 44
III II II I 45 III II 46 III II 47 IV IV 48 IV IV IV II 49 IV IV IV
II 50 IV IV I 51 IV IV IV I 52 IV IV III I 53 IV IV IV II 54 IV IV
I 55 IV IV III II II II 56 III 57 III 58 II 59 I 60 IV IV IV II 61
II 62 III 63 III 64 IV IV II 65 IV 66 II 67 IV 68 II 69 II 70 IV IV
II 71 II 72 IV IV II 73 IV 74 IV IV I 75 III 76 III III 77 IV IV IV
III III III 78 IV IV IV II II II 79 IV IV IV III III III 80 II 81
IV IV IV III III III II 82 III 83 II II 84 III II 85 IV III 86 I I
III 87 II II II III 88 I 89 II II III 90 II II II 91 II II II 92 II
II II II 93 I 94 II 95 II 96 II 97 II III II III 98 IV 99 IV 100
III 101 III 102 IV 103 IV 104 IV 105 IV vorinostat III III III
Wortmannin IV IV IV III
Example 11: Cell Culture and Anti-Proliferative Assays (Cellular
IC.sub.50)
[0339] Representative human tumour cell lines used in the cellular
assays are breast cancer (BT-474, MCF7, MDA-MB-231, MDA-MB-436, and
MDA-MB-468), colon cancer (COLO 205 and HCT 116), glioblastoma (U87
MG), leukemia (HL-60, K-562, MOLT-4, MV-4-11, RPMI-8226, SUP-B15),
lung cancer (A549, NCI-H460, and NCI-H522), melanoma (A-375),
ovarian cancer (SK-OV-3), pancreases cancer (BxPC-3 and PANC-1),
prostate cancer (PC-3 and DU145), and renal cancer (A-498 and ACHN)
cell lines. Cell lines COLO 205, HCT 116, MOLT-4, MV-4-11, K-562,
RPMI8226, SUP-B15, U87MG, SK-OV-3, PC-3, DU-145, NCI-H460,
NCI-H522, A375, HepG2, SK-HEP1, BxPC-3 and PANC-1 were purchased
from ATCC and expanded using ATCC recommended media. Cell lines
MCF7, TB474, T-47D, MDA-MB-231, MDA-MB-468, MDA-MB-436, A549,
A-498, AHCN, and HeLa were also from ATCC. Cells were cultivated at
37.degree. C., 5% CO.sub.2 in media containing 10% FBS and 2 mM
glutamine. Cells were routinely monitored with MycoAlert.TM. PLUS
Mycoplasma Detection Kit (Lonza Walkersville, Inc.) to make sure
they were mycoplasma free. Antibiotics (100 U/mL of penicillin and
100 .mu.g/mL of streptomycin) were only added to the media used for
compound dilution and cells in 96-well assay plates. DMEM (4.5 g/L
glucose) and RPMI1640 were obtained from Biopolis Shared Facilities
(BSF), Singapore. For cellular assays, BxPC-3, PC-3, HCT 116, COLO
205, MV-4-11, RPMI8226, HL-60, SUP-B15, T-47D were cultivated in
RPMI1640; A-498, ACHN, DU145, MCF7, PANC-1, U-87MG, NCI-H460,
NCI-H522, HeLa, and A-375 were cultivated in DMEM; BT-474,
MDA-MB-231, MDA-MB-468, MDA-MB-436 in DMEM/F12 (1:1).
[0340] For a typical screening experiment, cells (100 .mu.L/well)
are inoculated into 96 well microtiter plates at plating densities
ranging from 2,000 to 30,000 cells/well depending on the doubling
time of individual cell lines and assay linearity. Clear 96-well
tissue culture plates (Corning #3596, or Nunc 167008) are used for
colorimetric assay and monitoring cell growth status, while white
96-well tissue culture plates (Corning #3917) are used for
luminescent and fluorescent assays. After cell inoculation, the
microtiter plates are incubated at 37.degree. C., 5% CO.sub.2, 95%
air and 100% relative humidity overnight (up to 24 h) prior to
addition of compounds for adherent cells. Suspension cells are
treated with test compounds immediately after cell inoculation.
[0341] Test compounds are serially diluted (3-fold or 4-fold,
8-concentration) using the same media and added to the plates (15
to 25 .mu.L/well). After 72 h of cultivation, the plates are
assayed for cell cytotoxicity/viability by using the following two
methods. For selected compounds, IC.sub.50s after 24 h and 48 h of
drug treatment were also determined.
Sulforhodamine B (SRB) Method
[0342] Cells in plates are fixed with cold trichloroacetic acid
(TCA) (50% w/v in DI water, 1/4 volume of the medium in each well),
and incubated at 4.degree. C. for 1 h (adherent cells) or 2 h
(suspension cells). The plates are washed 5 times (tap
water.times.3, DI water.times.2) and air-dried. SRB solution 0.4%
(w/v) in 1% acetic acid is added to each well (60 .mu.L/well) and
the plates are incubated at room temperature for 30 min, then
washed with 1% acetic acid solution 4 times, and air-dried. The
bound dye is dissolved by addition of Tris base solution (10 mM,
100 .mu.L/well) and the absorbance is read at a wavelength of 515
nm on a BioTek Synergy H4 Hybrid Multi-Mode Microplate Reader.
CellTiter-Glo.RTM. Luminescent Cell Viability Assay
[0343] The plates are added CellTiter-Glo.RTM. Reagent (95
.mu.L/well) and read the luminescence on a BioTek H4 reader per
manufacture's protocol.
[0344] The raw data are processed using BioTek's Software Gen5
(v2.03.01) to generate inhibitory IC.sub.50 values. Vorinostat and
GDC-0941 are used as positive control.
[0345] IC.sub.50 is defined as the concentration of compound
required for 50% inhibition of cell vs non-treated. IC.sub.50 data
are shown in Tables 4 and 5 below.
Definition of the potency is as follows: "I", IC.sub.50>10
.mu.M. "II": 2 .mu.M<IC.sub.50.ltoreq.10 .mu.M. "III": 0.5
.mu.M<IC.sub.50.ltoreq.2 .mu.M. "IV": IC.sub.50.ltoreq.0.5
.mu.M
TABLE-US-00007 TABLE 4 In Vitro Cellular IC.sub.50 (.mu.M) EX
MV-4-11 K-562 MOLT-4 PC-3 MCF7 COLO 205 HCT 116 HepG2 1 IV III IV
III III III IV III 2 IV IV IV III III III III III 3 III II II II
III 4 III III 5 IV III II III III III 6 III II II II II II III 7
III III III III IV III III III 10 IV III II III II 29 IV III IV III
III III IV 30 IV II IV II II 31 IV III III II III III 37 IV III IV
II III II III III 40 III III III II III II III II 41 IV IV IV II
III III III III 43 IV IV IV III III III 44 IV III IV III III III
III 45 IV III IV III III III 46 IV IV IV III III III 47 III III IV
II III III 48 III III IV II III III III 49 III III IV II III III II
50 III III IV II III III II 51 IV III II III III III 52 III II II
III III 53 IV III III II III III III III 54 III II II II II 55 IV
III IV II III III IV III 56 III II II 57 I I I 58 III II II 59 III
I I 60 IV III III II II II III II 61 III I I 62 IV II III II III 63
III II II 64 III I I 65 I I I 66 III II II 67 III II II 68 III II
II 69 III II II 70 III II II 71 III I I 72 III III III 73 III II 74
III IV IV III III III III 75 IV II III III II 76 IV III IV III III
IV III 77 IV III IV II III III II 78 IV IV IV III III III III 79 IV
III IV III III III II 82 III I II I 83 II III II 84 II II I 85 III
III IV II III III 86 II III II 87 I II I 88 I II I 89 II III II III
II 90 II II II 91 II II II 92 II II II 93 III II II II II 94 III II
II 95 III II I 96 III II III II III III III III 97 III II II II 98
III III II III II II 99 III II II 100 IV IV IV IV III 101 IV IV IV
III III 102 IV III III 103 IV IV III 104 IV III III 105 IV IV III
III GDC-0941 III I IV III IV III III I GDC-0980 I II II IV
vorinostat III III III II III III III III Sorafenib IV II II II II
III II
TABLE-US-00008 TABLE 5 In Vitro Cellular IC50 (.mu.M)* for
representative compound EX 1, 2, 7, 37, 41, 46, 55, 78, 98, and 100
Cell Lines Cancer Panel 1 2 7 37 41 46 55 78 98 100 GDC-0941
Vorinostat Sorafenib BT474 Breast Cancer III III IV III III III IV
II MCF7 Breast Cancer III III IV III III III III III III IV IV III
II MDA-MB-231 Breast Cancer III III II III II I II MDA-MB-436
Breast Cancer III III II III II III II MDA-MB-468 Breast Cancer III
III IV III III II III 4T1 Breast cancer (mouse) III III II III IV
III III II II COLO 205 Colon Cancer III III III II III III III II
III III HCT 116 Colon Cancer IV III III III III IV III IV II III II
U138MG Glioblastoma III III III III III III II II U87 MG
Glioblastoma III II II II II III III III II II II HuH-7 HCC III IV
III III IV III III II SK-HEP1 HCC III III III III III III III III
II III II HCCLM3 HCC II III II II II III II II HepG2 HCC III III
III III III III III II III I III II PLC/PRF/5 HCC III III III II
III II III II HEL9217 Leukemia III IV IV IV IV II III HL-60
Leukemia IV IV III III IV III III K-562 Leukemia III IV III III IV
IV III IV III I III II MOLT-4 Leukemia IV IV III IV IV IV IV IV IV
IV III II MV-4-11 Leukemia IV IV III IV IV IV IV IV III IV III III
IV RPMI-8226 Leukemia IV III III III IV II III SUP-B15 Leukemia IV
IV IV IV IV II III NCI-H460 Lung Cancer III III III III IV IV III
II NCI-H522 Lung Cancer III III II III II III III III I II II A549
Lung Cancer III III III III IV IV III II Pfeiffer Lymphoma IV IV IV
IV IV III IV IV IV III II Ramos Lymphoma IV IV III IV IV IV IV II
III Daudi Lymphoma IV IV IV IV IV IV IV Raji Lymphoma III IV IV IV
IV II III II A375 Melanoma III III II III III III III III BxPC-3
Pancreases III III III III III III II PANC-1 Pancreases III II II
II II II III II PC-3 Prostate Cancer III III III II II III II III
II III II II ACHN Renal Cancer III III II III III III III III A-431
Skin cancer II III III III III IV II *"I":, IC.sub.50 > 10
.mu.M. "II": 2 .mu.M < IC.sub.50 .ltoreq. 10 uM. "III": 0.5
.mu.M < IC.sub.50 .ltoreq. 2 .mu.M. "IV": IC.sub.50 .ltoreq. 0.5
.mu.M. HCC = Hepatocellular carcinoma
Example 12: Western Blot Analysis
[0346] Cells are cultivated and treated with test compounds as
described in the above Cell Culture and Anti-Proliferative Assays
section (Example 11). Cells are washed with cold DPBS twice and
cooled on ice and treated with lysis buffer [50 mM Tris (pH7.4),
2.5 mM .beta.-glycerophosphate, 150 mM NaCl, 1% NP-40, 0.5% sodium
deoxycholate, 0.1% SDS, 2 mM sodium orthovanadate, 10 mM sodium
fluoride, 1 mM EDTA and freshly added protease inhibitor PMSF (0.1
mM) and protease inhibitor cocktail (Cat #03969, Nacalai Tesque,
Inc. or Cat. #539134, Calbiochem). Lysates are cleared at
20,000.times.g 20.times.2 min, and protein concentrations are
determined using BCA Protein Assay K (Cat #71285-3, Novagen, USA).
Proteins in cell lysates are resolved by SDS-PAGE and transferred
to PVDF and probed with appropriate primary and secondary
antibodies. Histone H3 (acetyl K9) (Cat #9649), Histone H3 (Cat
#9715), Acetyl-.alpha.-Tubulin (Lys40) (Cat #5335), .alpha.-Tubulin
(Cat #2125), pAkt (Ser473) (Cat #4060), pAkt (Thr308) (Cat #4056,
#2965), anti-Akt (pan) (Cat #4685), pS6 ribosomal protein
(Ser240/244) (Cat #5364), S6 ribosomal protein (Cat #2217), pS6
ribosomal protein (S240/244), Phospho-PRAS40 (Thr246) (Cat #2997),
p-mTOR (S2448) (Cat #5536), pErk1/2 (T202/y204) (Cat#4376),
Phospho-4E-BP1 (Thr37/46) (Cat #2855), p70S6 Kinase (Cat #2708),
Phospho-p70 S6 Kinase (Thr389) (Cat #9234), Phospho-p70S6 Kinase
(Thr421/Ser424) (Cat #9204), Phospho-p70S6 Kinase (Ser371) (Cat
#9208), pPDK1 (Ser241) (Cat #3438), and HRP-linked anti-rabbit IgG
(Cat #7074) antibodies were purchased from Cell Signaling
Technology, Inc., USA. Anti-(3-actin (Cat # ab8227) was from Abcam,
and anti-GAPDH-HRP (Cat # sc-25778-HRP) was from Santa Cruz
Biotechnology, Inc. The protein bands are detected using Pierce ECL
Western Blotting Substrate (Cat #3229) and captured using FUJI
Super RX-N films which are subsequently scanned and analyzed using
ImageJ (1.47V) software. Representative Western blot images are
shown in FIG. 15 and analyzed results are presented by FIG. 17 to
20.
[0347] In FIG. 15, the inhibition of HDACs and PI3k-Akt-mTOR
pathway in PC-3 cells are shown. PC-3 cells were treated with test
compounds at the following concentrations (all contain 0.1% DMSO)
for 24 h at 37.degree. C., and then processed according to section
of Western Blot Analysis.
TABLE-US-00009 Lane Sample 1 DMSO (0.1%) 2 Vorinostat_10.mu.M 3
GDC-0941_1uM 4 EX1_1.mu.M 5 EX1_10.mu.M 6 EX2_1.mu.M 7 EX2_10.mu.M
8 Insulin (100 .mu.g/mL), 30 min A EX3_10.mu.M B EX6_10.mu.M C
EX37_10.mu.M D EX40_10.mu.M E EX41_10.mu.M F EX7_10.mu.M G
EX97_10.mu.M
Hyperacetylation of histone 3 (Lys 9) and .alpha.-tubulin were
observed for compound examples tested (FIGS. 15A, 15B, 15C and 15D)
and the hyperacetylation effect is dose-dependent as demonstrated
by both EX1 and EX2 (FIG. 15A). The tested examples also inhibited
PI3K-Akt-mTOR pathway with a broad range of activities. Except EX40
and EX41, they all inhibited phosphorylation of Akt (Ser473) or
activity of mTORC2 (FIGS. 15A and 15B). They also inhibited
phosphorylation of S6(Ser240/244) or activity of mTORC1 (with
exception of EX2, EX40 and EX41, FIGS. 15C and 15D). DMSO (0.1%)
was used as blank control for protein phosphorylation and
normalized as 100% when analyzed by ImageJ. PC-3 cells were also
treated with insulin (100 .mu.g/mL) for 30 min and both pAkt and
pS6 phosphorylations were significantly enhanced. The gels were
digitized and analyzed by ImageJ, see FIG. 17 to 20 for
details.
[0348] In FIG. 16, the modulations of PI3K-AKT-mTOR Pathway in MCF7
cells are shown. MCF7 cells were serum starved overnight and
treated with test compounds for 2 h including 30 min of insulin
stimulation (20 .mu.g/mL). V=vehicle DMSO (0.1%) as blank control,
and its phosphorylation level was normalized to 100% in ImageJ
analysis. The gels were digitized and analyzed by ImageJ; see FIGS.
17 to 20 for details.
[0349] In FIG. 17, the hyperacetylation of histone 3 (Lys 9) due to
inhibition of HDACs is shown. FIG. 17A shows PC-3 cells treated
with test compounds at above indicated concentrations (FIG. 15) for
24 h. The graph represents results from two Western Blot analyses.
Vorinostat (10 .mu.M) was used as positive control, and its
acetylation level was normalized to 100% in ImageJ analysis. FIG.
17B shows MCF7 cells that were serum starved overnight and treated
with test compounds for 2 h including insulin (20 .mu.g/mL)
stimulation in last 30 min for all samples. Vehicle DMSO (0.1%) was
used as blank control, and its acH3 level was normalized to 1. Y
Axis is expressed as Mean.+-.SD if applicable.
[0350] In FIG. 18, hyperacetylation of .alpha.-tubulin due to
inhibition of HDAC6 is shown. FIG. 18A shows PC-3 cells treated
with test compounds at above indicated concentrations (FIG. 15) for
24 h, Figure the graph represents results from two Western Blot
analyses. Vorinostat (10 .mu.M) was used as positive control, and
its acetylation level was normalized to 100% in ImageJ analysis.
FIG. 17B. shows MCF7 cells that were serum starved overnight and
treated with test compounds for 2 h including insulin (20 .mu.g/mL)
stimulation in last 30 min for all samples. Vehicle DMSO (0.1%) as
blank control, and its acetylation level was normalized to 1. Y
Axis is expressed as Mean.+-.SD if applicable.
[0351] In FIG. 19, the modulation of PI3K-AKT-(mTOR) Pathway: p-Akt
(Ser473) level and activity of mTORC2 are shown. FIG. 19A shows
PC-3 cells treated with test compounds at above indicated
concentrations (FIG. 15) for 24 h, the graph represents results
from two Western Blot analyses. Vehicle DMSO (0.1%) was used as
blank control, and its phosphorylation level was normalized to 100%
in ImageJ analysis. pAkt level was significantly enhanced in
insulin (100 .mu.g/mL, 30 min) treated cells. FIG. 19B shows MCF7
cells serum starved overnight and treated with test compounds for 2
h including insulin stimulation (20 .mu.g/mL) in last 30 min for
all samples. Vehicle DMSO (0.1%) was used as blank control, its
phosphorylation level was normalized to 100%. Y Axis is expressed
as Mean.+-.SD if applicable.
[0352] In FIG. 20, the modulation of PI3K-AKT-(mTOR) Pathway:
mTORC1 activity is shown. MCF7 cells were serum starved overnight
and treated with test compounds for 2 h including insulin
stimulation (20 .mu.g/mL) in last 30 min for all samples. Vehicle
DMSO (0.1%) as blank control, its phosphorylation level was
normalized to 100%. In FIG. 20A, p-P70S6K (Thr389)/p-P85S6K
(Thr412) level in MCF7 cells is shown. In FIG. 20B, p-S6
(Ser240/244) level in MCF7 cells is shown. In FIG. 20C, the
p-4E-BP1 (Thr37/46) level in MCF cells is shown. In FIG. 20D, PC-3
cells were treated with test compounds at above indicated
concentrations (FIG. 15) for 24 h. pS6 level was enhanced in
insulin (100 .mu.g/mL, 30 min) treated cells. Y Axis is expressed
as Mean.+-.SD if applicable.
Example 13: Caspase Activity Assays
[0353] Cells are cultivated and treated with test compounds in
96-well plates as described in the above Cell Culture and
Anti-Proliferative Assays section. Caspase assay buffer [100 mM
HEPES (pH7.5), 200 mM NaCl, 4 mM EDTA, 0.1% CHAPS, and freshly
added 5 mM DTT and 50 .mu.M of Caspase substrate Z-DEVD-R110 or
(Z-Asp-Glu-Val-Asp).sub.2-Rhodamine 110 (Cat #M-2615, Bachem,
Switzerland)] is added to cells (100 .mu.L/well) and the plates are
incubated at room temperature and monitored for the Caspase
activity on a BioTek Synergy H4 reader (excitation: 496/9 nm,
emission: 521/9 nm). The incubation time can be extended up to
overnight (18 h) if the Caspase activity is low. Staurosporine is
used as positive control. Representative results are shown in FIG.
21.
[0354] FIG. 21 shows the caspase activity of tested examples. Cells
were treated with EX1, EX2, GDC-0941, vorinostat and staurosporine
(as positive control) and caspase activity was monitored at
different time points. FIG. 21A shows MV-4-11 cells, caspase
activity was monitored at 6, 24, 48 and 72 h, and maximal activity
was found at 24 h. Both EX2 and EX1 are more potent than vorinostat
in terms of induction of caspase activity. GDC0941 showed very weak
activity and potency. EC.sub.50=0.52, 0.50, 1.16, and 12.7 .mu.M
for EX1, EX2, vorinostat and GDC-0941, respectively. FIG. 21B shows
PC-3 cells monitored at 24, 48 and 72 h time points. Maximal
caspase activity was found at 48 h. Both EX1 and EX2 are more
potent than vorinostat in terms of induction of caspase activity.
GDC-0941 showed very weak activity in both tumor cells. Y Axis is
expressed as Mean.+-.SD if applicable.
Example 14: Cell Viability/Cytotoxicity Assay
[0355] Cells are cultivated and treated with test compounds in
96-well plates as described in the above Cell Culture and
Anti-Proliferative Assays section, the content of dead cells and
viable cells are measured using CytoTox-Glo.TM. Cytotoxicity Assay
kit (Promega) per manufacture's protocol. Briefly, CytoTox-Glo.TM.
Cytotoxicity Assay Reagent (48 .mu.L/well) is added to the cells in
96-well plate and the luminescent signals are recorded using a
BioTek Synergy H4 reader, then Lysis Reagent (48 .mu.L/well) is
added and the luminescent signals derived from both dead and viable
cells are recorded. Viability can be calculated by subtracting the
luminescent signal resulting from experimental cell death from
total luminescent values. Staurosporine is used as positive
control. Representative results are shown in FIG. 22.
[0356] FIG. 22 shows the compound induced death of MV-4-11 cells.
Cells were treated with EX1, EX2, GDC-0941, vorinostat and
staurosporine (as positive control) and CytoTox-Glo.TM.
Cytotoxicity Assay kit was used to assess the cell death. Fold of
cell death of treated vs untreated was monitored at different time
points. Maximal cell death occurred at 48 h after addition of test
compounds. FIG. 22A shows EX1, compound example 1. FIG. 22B shows
EX2, compound example 2. FIG. 22C shows GDC-0941. FIG. 22D shows
cell death at 48 h for EX1, EX2, GDC-0941, and vorinostat. The cell
death potency EC.sub.50 was estimated as 0.19, 0.069, 0.58 and 2.75
.mu.M for EX1, EX2, vorinostat and GDC-0941, respectively. Y Axis
is expressed as Mean.+-.SD if applicable.
Example 15: Microsomal Stability
[0357] GIBIO pooled human liver microsomes (HLM) (Cat # HMMCPL),
mouse liver microsomes (MLM) (Cat # BCMCPL), and rat liver
microsomes (RLM) (Cat # RTMCPL) were purchased from Life
Technologies. The incubations consisted of test compound (5 .mu.M)
or control compounds (verapamil and dextromethorphan), 0.5 mg/mL of
microsomes, 3.3 mM MgCl.sub.2, 1.3 mM .beta.-NADPH, and 100 mM
potassium phosphate buffer (pH 7.4). Samples are incubated for 30,
45, or 60 min. Reaction is terminated with ice-cold acetonitrile
0.3% formic acid. Samples are subsequently centrifuged at 4.degree.
C. for 15 min at 20,000.times.g. The supernatant is analysed by
LC-MS. Representative results are shown in Table 6.
TABLE-US-00010 TABLE 6 Microsomal Stability EX HLM MLM RLM 1 101%
96% 70% 2 99% 99% 57% 4 36% 42% 5 89% 133% 22% 6 88% 89% 88% 10 87%
74% 29 97% 75% 9% 31 90% 53% 25% 37 72% 73% 36% 41 100% 93% 42% 43
97% 96% 54% 44 84% 84% 53% 45 105% 78% 45% 46 93% 89% 50% 51 86% 9%
29% 52 87% 102% 42% 53 100% 84% 43% 54 94% 48% 54% 55 90% 83% 103%
60 95% 71% 82% 82 83% 64% 89 87% 70% 70% 98 92% 83% 91% Verapamil
34% 24% 12% Dextromethorphan 65% 19% 5%
[0358] Compound in vitro metabolic stability was assayed using
liver microsomes (LM) at 0.5 mg/mL of proteins, incubated for 30
min (mouse, MLM), 45 min (rat, RLM), 30 or 45 min (human, HLM). %
of remaining parent compound was measured using LC-MS. Both
verapamil and dextromethorphan were used as positive controls.
Example 16: Pharmacokinetics (PK)
[0359] All animal studies were done as per approved protocols by
the Institutional Animal Care and Use Committee at the Biological
Resource Centre (BRC) in Singapore. BALB/c mice (8-12 week old,
BRC, Biopolis, Singapore) were dosed i.v., p.o. and i.p. with a
variety of formulated solutions or suspensions of compound
examples. Blood was collected after serial bleeding and
centrifuged, and the plasma was frozen at -80.degree. C. Tissues
(e.g., livers, lungs, and kidney) were snap frozen in dry ice or
liquid nitrogen and kept at -80.degree. C. until analysis. The
plasma samples were added internal standard carbamazepine (CBZ) and
processed as described previously (Jayaraman, et al. Drug Metab.
Dispos. 2011, 39, 2219-2232). Quantitative analysis was carried out
on a Waters 2795 separations module equipped with a Waters 2996
Photodiode Array (PDA) detector and micromass Quattro micro mass
spectrometer. Sample was resolved on Phenomenex Luna C18(2),
2.0.times.50 mm column with a SecurityGuard Cartridge (C18
4.times.2.0 mm) at a flow of 0.5 mL/min with a 6-min gradient (x to
95% of B, solvent A, ultrapure water with 0.1% of formic acid (FA),
solvent B, methanol with 0.1% of FA, x is selected from 5 to 50)
and data were acquired using multiple reaction monitoring and
quantified by QuanLynx in MasLynx software (V 4.1, Waters Inc.). PK
parameters were estimated using Microsoft Excel 2010 based on the
PK equations defined by Summit PK website
(http://www.summitpk.com/equations/equations.htm). Both area under
curve calculations and multi-exponential curve stripping were used.
The method can deliver comparable results as WinNonlin (Pharsight,
Mountain View, Calif.) for our previous data.
Example 17: In Vivo Pharmacodynamics (PD) and Efficacy Studies
[0360] All animal studies were done as per approved protocols by
the Institutional Animal Care and Use Committee at the Biological
Resource Centre (BRC) in Singapore. Female BALB/c nude mice (7 and
10 weeks of age, BRC, Biopolis, Singapore) or female NCr nude mice
(5-6 and 7-9 weeks of age, InVivos Pte Ltd, Singapore) were
inoculated in the right flank with about 5.times.10.sup.6 of tumor
cells which were suspended in serum-free DMEM or RPMI1640 growth
medium and Matrigel (Cat. No: 354234, Corning Discovery Labware)
(1:1) and injected in a total volume of 100 to 150 .mu.L. Tumor
were measured using a digital caliper and tumor volumes was
estimated by using the formula: tumor
volume=length.times.width.sup.2.times.0.5. Tumor growth inhibition
(TGI %)=[1-(T.sub.t-T.sub.0)/(C.sub.t-C.sub.0).times.100, C.sub.0
and C.sub.t are the mean tumor volumes for control group (vehicle)
on day 0 and day t, respectively; T.sub.0 and T.sub.t are the mean
tumor volumes for treatment group on day 0 and day t, respectively.
All statistics conducted were done using GraphPad Prism (v4.00 or
v6.04, GraphPad Software Inc.), two-tailed unpaired t Test was used
for comparing two groups, and one way ANOVA followed by Dunnett's
Multiple Comparison Test was used for comparing three and more
groups.
Example 18: Target Modulation
In PC-3 Prostate Cancer Xenograft
[0361] PC-3 tumor bearing BALB/c nude mice were orally dosed with
vehicle, vorinostat (200 mg/kg), EX1 (150 mg/kg) and EX78 (100
mg/kg). Blood samples, tumors and other tissues were collected for
PK/PD studies at the indicated time points (two mice each time
point). Hyperacetylation of H3 in PC-3 tumors was confirmed by
Western blot analyses of tumors of the EX1 and EX78 treated mice,
vorinostat was used as positive control (FIG. 23).
[0362] FIG. 23 shows histone hyperacetylation in PC-3 tumors. FIG.
23A shows a Western blot analysis of tumor tissues. The lanes and
the concentrations used were as follows:
TABLE-US-00011 Lane Sample 1/2 Vehicle at 3 h 3/4 Vorinostat at 3 h
5/6 EX1 at 1 h 7/8 EX1 at 2 h 9/A EX1 at 3 h B EX78 at 4 h
Significant histone hyperacetylation in PC-3 tumor was observed in
treated mice. FIG. 23B shows the digitized and normalized Western
blot analysis results. Y Axis is expressed as Mean.+-.SEM if
applicable.
In MV4-11 Acute Myeloid Leukemia Xenograft
[0363] MV4-11 tumor bearing mice were also treated with EX2 via
intravenous (IV) (50 mg/kg), intraperitoneal (IP) (100 mg/kg) and
per orem (PO) (150 mg/kg) routes, EX78 via both IV (25 mg/kg) and
PO (100 mg/kg) routes for Pharmacodynamic (PD) assessment. All
three routes of administration of EX2 result in hyperacetylation of
H3 in MV4-11 tumors (FIG. 24). EX78 also induced hyperacetylation
of H3 via both routes of administration.
[0364] FIG. 24 shows Histone Hyperacetylation in MV4-11 tumors.
BALB/c nude mice bearing MV4-11 tumors which were dosed with
vehicle [DMSO/PEG400/sterile water (10:40:50)], EX2 and EX78.
Tumors were collected at the indicated time points. FIG. 24A shows
Western blot analyses of tumor tissues.
[0365] The lanes and the concentrations used were as follows:
TABLE-US-00012 Lane Sample 1 Vehicle at 3 h, PO 2 EX2 at 1 h, PO 3
EX2 at 2 h, PO 4/5/6 EX2 at 3 h, PO 7 EX2 at 2 h, IV 8 EX2 at 3 h,
IV 9 EX2 at 2 h, IP A EX78 at 1 h, IV B EX78 at 2 h, PO C EX78 at 3
h, PO D EX78 at 4 h, PO
Significant histone hyperacetylation in MV4-11 tumor was observed
in treated mice. FIG. 24B shows the digitized and normalized
Western blot analysis results. Y Axis is expressed as Mean.+-.SEM
if applicable.
Example 19: Efficacy
In NCr Nude Mice HepG2 Xenograft Model
[0366] Female NCr nude mice (CrTac:NCr-Foxn1.sup.nn, 5 weeks of
age, InVivos Pte Ltd, Singapore) were inoculated in the right flank
with 6.times.10.sup.6 of HepG2 cells. When the HepG2 tumor size was
275 mm.sup.3 in average (13 days after implantation), the mice were
randomized and dosed orally with vehicle, EX2 (150 mg/kg) and
sorafenib tosylate (98 mg/kg) for four weeks (QD.times.5 per week).
The mice of vehicle group were euthanized due to the tumor burden
on day 18 after last dose of 3.sup.rd cycles. EX2 demonstrated
significant tumor inhibition with TGI=96% on day 18 (after last
dose) (FIG. 25). Reference sorafenib tosylate (98 mg/kg) was also
effective but with TGI=71% (day 18).
[0367] FIG. 25 shows efficacy in NCr nude mice HepG2 xenograft
model. HepG2 tumor-bearing female NCr nude mice were treated with
either vehicle or EX2 (150 mg/kg) from Day 0 with mean tumor volume
about 275 mm.sup.3 for four cycles: 5-day-on-2-day-off (QD.times.5
per week or per cycle). FIG. 25A shows significant tumor growth
inhibition (TGI) was achieved from day 4. TGI=96%, p=0.0034 on day
18 after last dose of 3.sup.rd cycle. The treatment continued to
4.sup.th cycle while the mice of vehicle group were euthanized due
to the tumor burden on day 18. FIG. 25B shows that EX2 was well
tolerated in NCr nude mice, no significant toxicity was observed at
this dose level. Y Axis is expressed as Mean.+-.SEM if
applicable.
In CB17 Scid Mice HepG2 Xenograft Model
[0368] As EX2 showed excellent antitumor activity in HepG2 tumor
bearing NCr nude mice, it was further evaluated with dose repose in
CB17 scid mice. Female C.B-17 scid mice
(C.B-Igh-1.sup.b/IcrTac-Prkdc.sup.scid, 5 weeks of age, InVivos Pte
Ltd, Singapore) were inoculated in the right flank with
5.times.10.sup.6 of HepG2 cells. When the HepG2 tumor size was
about 240 mm.sup.3 in average, tumor-bearing mice were randomized
(5 mice per group) and dosed orally with vehicle, EX2 (150, 75 and
37.5 mg/kg) and sorafenib tosylate for four weeks (QD.times.5 per
week) on day 0. EX2 demonstrated significant tumor inhibition in a
dose-dependent manner. All dose levels were well tolerated and
significant tumor growth delay was achieved (FIG. 26).
[0369] In FIG. 26, Efficacy in CB17 scid mice HepG2 xenograft model
is shown. HepG2 tumor-bearing CB17 scid mice were treated with
vehicle, EX2 (150, 75 and 37.5 mg/kg) and sorafenib tosylate (100
mg/kg QD.times.5.times.2 then 80 mg/kg, QD.times.5.times.2) for 4
weeks (QD.times.5 per week) from Day 0 with mean tumor volume about
240 mm.sup.3. FIG. 26A shows that significant tumor growth
inhibition (TGI) was achieved from day 4. After last dose of
4.sup.th cycles, on day 26, TGI=117%, 82%, 38% and 98% for EX2
(150, 75 and 37.5 mg/kg) and sorafenib tosylate, respectively. FIG.
26B shows that on day 26, tumor size of treated group was
significantly smaller than vehicle group, with p<0.01 for EX2
150 and 75 mg/kg groups and sorafenib group, but p>0.05 for 37.5
mg/kg group. FIG. 26C shows that EX2 was well tolerated at all dose
levels, no significant body weight (BW) loss. Vehicle group has BW
loss due to increasing tumor burden. Sorafenib tosylate (100 mg/kg)
was not well tolerated; its dose was reduced to 80 mg/kg in the
3.sup.rd and 4.sup.th cycles. FIG. 26D shows that tumor size was
normalized against the initial value (as 100%). After last dose
(day 25), the tumor started re-grow after a long period of growth
delay, but EX2 at 150 mg/kg seemed more effective than sorafenib. Y
Axis is expressed as Mean.+-.SEM if applicable.
In NCr Nude Mice HuH-7 Xenograft Model
[0370] Female NCr nude mice (CrTac:NCr-Foxn1.sup.nn, 8 weeks of
age, InVivos Pte Ltd, Singapore) were inoculated in the right flank
with 6.2.times.10.sup.6 of HuH-7 cells. When the HuH-7 tumor size
was about 103 mm.sup.3 in average, the mice were randomized and
dosed orally with vehicle [DMSO/Solutol.RTM. HS15/sterile water
(10:36:54)] and EX2 (150 mg/kg) for two weeks (QD.times.5 per week,
5 mice per group), respectively. EX2 demonstrated good tumor growth
inhibition with TGI=102% on day 12 (after last dose) and again EX2
was well-tolerated at this dose level (maximum body weight loss was
less than 5% vs vehicle group). The experiment was repeated with
large and well-established tumors. When the HuH-7 tumor size was
about 363 mm.sup.3 in average, the mice were randomized (5 mice per
group) and dosed orally with vehicle [NMP/Solutol.RTM. HS15/sterile
water (10:36:54)] and EX2 (150 mg/kg). EX2 demonstrated good tumor
growth inhibition with TGI=88% (p=0.0016) after one cycle of
treatment (QD.times.5 per week) on day 6 and TGI=67% (p=0.0082)
after two cycles of treatment (QD.times.5.times.2) on day 12 (FIG.
27).
[0371] In FIG. 27, the efficacy in NCr nude mice HuH-7 xenograft
model is shown. HuH-7 tumor-bearing NCr nude mice were treated with
either vehicle or EX2 (150 mg/kg) from Day 0 (QD.times.5 per week
or per cycle, or 5-day-on-2-day-off). FIG. 27A shows good tumor
growth inhibition with TGI=102% on day 12 after treatment of mice
(mean tumor volume 103 mm.sup.3 on day 0) with EX2
(QD.times.5.times.2). FIG. 27B shows that EX2 was also effective on
well-established tumors (mean tumor size 363 mm.sup.3 on day 0)
after one cycle of treatment with TGI=88% (p=0.0016) and TGI=67%
(p=0.0082) on day 12. The tumor size was normalized against the
initial volume (day 0 as 100%) in FIG. 27B. Y Axis is expressed as
Mean.+-.SEM if applicable.
In 4T1 Mouse Metastatic Breast Cancer Model
[0372] Female NCr nude mice (CrTac:NCr-Foxn1.sup.nn, 13 weeks of
age, InVivos Pte Ltd, Singapore) were implanted 1.1.times.10.sup.6
of 4T1 (ATCC.RTM. CRL-2539.TM.) cells in fourth mammary fat pad.
When 4T1 tumor size was 70-74 mm.sup.3 in average (5 days post
tumor implantation), tumor-bearing mice were randomized (n=5 per
group) and dosed orally with vehicle and EX2 (150 mg/kg) for three
cycles (QD.times.5, 5 day-on-1 day-off per cycle) day 0, day 16 was
the last dose of the 3 cycles. EX2 demonstrated significant tumor
inhibition with TGI=53% on day 17, p=0.0063 (FIG. 28).
[0373] In FIG. 28, the efficacy in 4T1 mouse metastatic breast
cancer model is shown. Female NCr nude mice bearing 4T1 tumor was
treated with vehicle and EX2 for three cycles (QD.times.5, 5
day-on-1 day-off per cycle, 5 mice per group). In FIG. 28A, the
tumor growth curve is shown. In FIG. 28B the tumor size on day 17
is shown, p=0.0063. Y Axis is expressed as Mean.+-.SEM if
applicable.
In NCI-H460 Lung Cancer Xenograft Model
[0374] Female C.B-17 scid mice
(C.B-Igh-1.sup.b/IcrTac-Prkdc.sup.scid, 7 weeks of age, InVivos Pte
Ltd, Singapore) were inoculated in the right flank with
6.8.times.10.sup.6 of NCI-H460 cells. When the tumor sizes were
between 150 and 160 mm.sup.3 in average, the mice were randomized
and dosed orally with vehicle and EX2 (150 mg/kg) for two weeks
(QD.times.5 per week) on day 0. EX2 demonstrated significant tumor
growth inhibition with TGI=46% (p=0.0292) on day 12 after two
cycles of treatment. EX2 was also well tolerated at this dose level
(FIG. 29).
[0375] In FIG. 29, the efficacy in NCI-H460 lung cancer xenograft
model is shown. Female NCI-H460 tuynor-bearing CB17 scid mice were
dosed orally with vehicle and EX2 (150 mg/kg) for two weeks
(QD.times.5 per week). FIG. 29A shows that EX2 demonstrated
significant tumor growth inhibition with TGI=46% (p=0.0292) on day
12 after two cycles of treatment. FIG. 29B shows that EX2 was also
well tolerated at this dose level. Y Axis is expressed as Mean -SEM
if applicable.
In MV4-11 Xenograft Model
[0376] Female BALB/c nude (C.Cg/AnNTac-Foxn1.sup.nn [cc]NE9, 5
weeks of age, InVivos Pte Ltd, Singapore) were inoculated in the
right flank with 11.times.10.sup.6 of MV4-11 cells. When the tumor
size was 173 mm.sup.3 in average, the mice were randomized (6 mice
per group) and dosed orally with vehicle and EX2 (150 and 75 mg/kg)
for three weeks (QD.times.5 per week) on day 0. From day 12 to day
20, EX2 demonstrated average TGI=52% (p<0.05) for 150 mg/kg
group (two groups, one non-treatment related death on day 6, final
n=11), but TGI=23% (p>0.05) for 75 mg/kg group (FIG. 30).
[0377] In FIG. 30, the efficacy in MV4-11 xenograft model is shown.
Tumor-bearing female BALB/c nude mice were dosed orally with
vehicle and EX2 (150 and 75 mg/kg) for three weeks (QD.times.5 per
week). FIG. 30A shows the tumor growth curve: tumor size was
normalized against the initial value (day 0 as 100%), EX2
demonstrated significant tumor inhibition with average TGI=51%
between day 12 and day 20 (p<0.05) for 150 mg/kg group, but 75
mg/kg was not significantly effective. FIG. 30B shows the tumor
size on day 20, TGI=51% (p<0.05) and 20% (p>0.05) for 150 and
75 mg/kg, respectively. Y Axis is expressed as Mean.+-.SEM if
applicable.
Example 20: Summary
[0378] The compounds as defined above demonstrated inhibitory
activities against HDAC enzymes and PI3K kinases (Table 3) and
anti-proliferative activities against a variety of human tumour
cell lines (Tables 4 and 5). Most of the compound as defined above
demonstrated good drug-like properties, that is, in vitro metabolic
stability, solubility and desirable lipophilicity (Table 6).
Selected compounds also showed activity against multi-targets in
tumor cells (FIG. 15 and FIG. 16), i.e., hyperacetylation of
histones (FIG. 17) and .alpha.-tubulin (FIG. 18) due to inhibition
of HDACs; PI3K-AKT-mTOR pathway: reduction of phosphor-Akt (Ser473)
or inhibition the activity of mTORC2 (FIG. 19), and reduction of
phospho-P70S6K (Thr389)/phospho-P85S6K (Thr412), phospho-S6
(Ser240/244) and phospho-4E-BP1 (Thr37/46) or inhibition of the
activity of mTOCR1 (FIG. 20). These compounds also induced cell
apoptosis in PC-3 cells and MV-4-11 cells (FIG. 21), cell death in
MV-4-11 cells (FIG. 22), much more efficiently than PI3k inhibitor
GDC-0941 or HDAC inhibitor vorinostat.
[0379] These compounds also modulated biological drug targets in
tumor models. For example, EX1 and EX78 induced histone
hyperacetylation in PC-3 prostate tumors when orally dosed in
tumor-bearing mice (FIG. 23), EX2 and EX78 induced histone
hyperacetylation in MV4-11 xenograft tumors via different routes of
administration (FIG. 24). EX2 also demonstrated excellent antitumor
activity in HCC models, e.g., NCr nude mice HepG2 xenograft model
(FIG. 25) and CB17 scid mice HepG2 xenograft model (FIG. 26) as
well as HuH-7 HCC xenograft model (FIG. 27). Compound EX2 was also
demonstrated broad antitumor activity in a number xenograft models
when dosed orally: 4T1 mouse metastatic breast cancer model (FIG.
28), NCI-H460 lung cancer xenograft model (FIG. 29) and MV4-11
leukaemia xenograft model (FIG. 30).
INDUSTRIAL APPLICABILITY
[0380] The compounds as defined above may find a multiple number of
applications in which their ability to inhibit deacetylases, lipid
and protein kinases of the type mentioned above can be utilised.
For example the compounds as defined above may be used to inhibit
deacetylase and kinases, either separately or simultaneously. The
compounds may also be used in treating or preventing a condition or
disorder in a mammal in which inhibition of a deacetylase and/or a
protein kinase and/or co-factor thereof and/or via an unspecified
mechanism prevents, inhibits or ameliorates a pathology or a
symptomology of the condition. The condition or disorder is cancer,
angiogenic disorder or pathological angiogenesis, fibrosis,
inflammatory conditions, asthma, neurological disorders,
neurodegenerative disorders, muscle degenerative disorders,
autoimmune disorders, disorders of the blood or disorders of the
bone marrow. The compounds may be particularly useful in treating
cancer such as leukemia or myeloma, lymphoma, breast cancer, lung
cancer, hepatocellular carcinoma and other hypervascular tumors as
well as retinal angiogenic diseases. The compounds as defined above
may also have applications in inducing cell reprogramming for
generation of iPS cells.
[0381] It will be apparent that various other modifications and
adaptations of the invention will be apparent to the person skilled
in the art after reading the foregoing disclosure without departing
from the spirit and scope of the invention and it is intended that
all such modifications and adaptations come within the scope of the
appended claims.
* * * * *
References